Background: Malaria is a febrile illness caused by protozoans of the genes Plasmodium. It has global distribution, but the burden is higher in the Sub-Saharan Africa. To decrease this burden, rapid diagnostic tests are good alternative diagnostic methods in remote areas where there is no laboratory. Histidine-rich protein-2 based rapid diagnostic tests have played a vital role in improving malaria case surveillance and management particularly in Africa, where P. falciparum is predominant. However, their usefulness is threatened by the emergence of Pfhrp2/3 gene deletion. Hence, monitoring of the deletion status at local and national level is mandatory to make policy decisions regarding diagnostic approaches. Objective: The study was aimed to assess the P. falciparum rapid diagnostic tests target gene (pfhrp2/3) deletion at selected health facilities in Amhara region, Ethiopia. Methods: An institution-based cross-sectional study was conducted in 28 selected health facilities from January 2021 -2022 in Amhara region, Ethiopia among 1232 participants. A convenient sampling was implemented to recruit consenting participants. A Structured questionnaire was used to collect sociodemographic and clinical data. Capillary blood was collected to perform the laboratory diagnosis by rapid diagnostic tests, microscopy and Polymerase chain reaction. Data were entered into “EPI Info version 7” software and exported to SPSS version 20.0 for analysis. Descriptive statistics was analyzed to explain study participants and rate of gene deletion. Results: From a total of 7529 screened individuals, 1177 P. falciparum, 427 P. vivax, 55 mixed infection were detected by light microscopy, while 1024, 432 and 60 participants were positive for P. falciparum, P.vivax and mixed infection by rapid diagnostic tests, respectively. From 1232 (1177 mono and 55 mixed infections) microscopy confirmed P.falciparum cases, 123 samples were suspected for Pfhrp2/3 gene deletion (i.e. microscopy positive but rapid diagnostic test negative). These 123 and 87 samples from discordant results (total 210) were recruited for molecular analysis. Of these, 158 (12.8%, 95%CI: 11.0-14.8) samples were with either one or both of Pfhrp2 and Pfhrp3 gene deletions. The proportion of false-negative Pfhrp2 rapid diagnostic test results due to pfhrp2/3 gene deletions was 106(9.0%, 95%CI: 8.4-11.8). Conclusions: The regional prevalence of Pfhrp2/3 gene deletion is above the WHO threshold rate (5%). It informs the national malaria program to make informed policy decision on rapid diagnostic tests. Keywords: RDT, P.falciparum, Histidine-rich protein 2/3, Gene deletion