Background: Blood transfusions are a life-saving medical procedure, yet they carry the risk of Transfusion-Transmitted infections, including Transfusion-Transmitted Malaria. This risk is heightened in Sub-Saharan Africa and Ethiopia due to a large pool of asymptomatic, low-parasitemia donors who evade conventional screening. The risk is compounded by the absence of routine malaria screening in Ethiopian blood banks and the limited sensitivity of available diagnostic methods. Examining the link between malaria and host genetic factors, such as ABO blood group, could support the development of targeted, resource-appropriate screening strategies. Objective: To determine the prevalence of malaria among blood donors and its association with ABO blood group variants using different diagnostic methods in Bahir Dar, Northwest Ethiopia. Methods: A facility-based cross-sectional study was conducted among 423 blood donors at five mobile blood collection sites in Bahir Dar, Northwest Ethiopia,from January–July 2026, using systematic random sampling. Three milliliters of Blood samples were used for screeningPlasmodium infection using BIOCREDIT pfLDH/pvLDH RDTand microscopy for 423 samples, and PCR (nested PCR and qPCR) on a sub-cohort of 221 samples. Sociodemographic and relateddata were collected using a structured questionnaire and analyzed using SPSS version 25 and RStudio software with descriptive statistics, Cohen's kappa, chi-square, and logistic regression (p<0.05 significant) Results: The malaria prevalence was 3.5% (15/423) by microscopy, 4% (17/423) by RDT, 4.5% (19/423) by composite RDT and microscopy, and 4.5% (9/202)by nested and qPCR.RDT and microscopy showed sensitivity of 56% and 52% respectively with a tie overall accuracy of 93.7%.P. vivaxwas predominant, with zero prevalence of P. malariae andP. ovale. Blood group O was most prevalent (47%; 199/423) and showed significantly higher malaria positivity (10.1%) and odds of infection (AOR=3.08, 95% CI: 1.14–9.11). Rural residence, malaria history, and travel to endemic areas independently increased infection odds, while Rh-positive status and ITN use were protective. Background: Malaria remains amajor public health problem in Ethiopia, including the Amhara Region. Although Plasmodium falciparum and Plasmodium vivax predominate, shifts in their relative distribution and recent reports of Plasmodium ovale indicate changing epidemiology. However, updated data on prevalence, species distribution, diagnostic performance, and associated factors remain limited. Routine diagnostics often miss low-density and mixed infections due to their low sensitivity.As well, the application of PCR is limited in the study area. Objective:The main aim of this study was to assess the prevalence, species distribution, diagnostic performance, and factors associated with Plasmodium infections among malaria-suspected individuals in Northwest Ethiopia. Methods:A health facility-based cross-sectional study was conducted from February to April 2026 in four study sites in Northwest Ethiopia. A total of 410 malaria-suspected individuals were enrolled using systematic random sampling. Sociodemographic, behavioral, and environmental data were collected using a structured questionnaire. A capillary blood sample was collected for Plasmodiumdetection and preparation of dried blood spots. Detection of Plasmodiumwas conducted using microscopy, BioCredit Malaria Pf/Pv pLDH rapid diagnostic test (RDT), and Polymerase Chain Reaction (PCR). Molecular detection and species identification were performed using multiplex real-time and nested PCR targeting the Plasmodium 18S rRNA gene.Data were entered using the statistical package for social science version 27 and analyzed in R version 4.5.1. Prevalence and species distribution were computed using descriptive statistics. Agreement between diagnostic methods was assessed using Cohen’s kappa (κ). Logistic regressionwas also used to identify factors associated with Plasmodium infection. P-value< 0.05 in the multivariate regression was taken as statistically significant. Results: Among 410 malaria-suspected participants, the overall prevalence of Plasmodiumspecies was 36.1% (148/410); positivity by RDT and by light microscopy was 23.2% (95/410) and 25.4% (104/410), respectively. Among 220 study participants,tested by PCR 52.7% (116/220) tested positive for Plasmodium species.Among RDT and microscopy-positive participants, PCR identified P. falciparum in 32/123 (26.0%), P. vivax in 24/123 (19.5%), mixed P. falciparum/P. vivax infections in 33/123 (26.8%), and P. malariae infection in 1/123 (0.8%). PCR detected a higher proportion of P. falciparum and mixed P. falciparum/P. vivax infections than RDT and microscopy.The prevalence of P. malariaeamong 220 study participants was1.7% (2/116). Most microscopy-positive infections were of low parasite density (60.6%; <1,000 parasites/µL), with median parasitemia of 2,505.0 parasites/µL for P. falciparum and 104.4 parasites/µL for P. vivax.. RDT and microscopy showed substantial agreement (κ = 0.688). Sensitivity and specificity of RDT compared withmicroscopy were 73.1% and 93.8%, respectively. Unimproved housing wall material, proximity to surface or stagnant water, nocturnal outdoor activity, and recent travel were independently associated with increased odds of Plasmodium infection. Conclusion and recommendations: Conventional diagnostic methods showed reduced sensitivity for low-density infections, while molecular methods revealed an additional Plasmodiumspecies profile, including P. malariae, missed by conventional methods. Infection risk was associated with household, environmental, and behavioral factors. Strengthening diagnostic quality, targeted molecular surveillance, housing improvement, drying stagnant water,and site-specific vector-control interventions are recommended to support malaria control in Northwest Ethiopia.