Genetics is the branch of biology that studies genes, heredity, and genetic variation in organisms Genetic testing is a medical test that analyzes DNA, the genetic material in cells, to identify changes or mutations in genes, chromosomes, or proteins. This study aimed to assess the knowledge, attitude, and challenges encountered by Healthcare Professionals in their practices related to genetic testing. The investigation took place at Bahir Dar Felege Hiwot Compressive Specialized Hospital, Tibebe Ghion Specialized Hospital, and Gambi Hospital, employing a cross-sectional study design. A total of 403 respondents were included, determined by utilizing the single population proportion formula. Primary data were collected through a semi structured questionnaire, and both descriptive and inferential statistics were applied to analyze the gathered information from healthcare professionals. Data management and analysis were carried out using SPSS 26 statistical software. In terms of knowledge, the mean score of the respondents was 16.29±2.89 out of 23. In multivariate logistic regression analysis, variables such as educational level, marital status, and age demonstrated statistical significance as independent factors influencing the knowledge of genetic testing among healthcare professionals. The mean attitude score among participants was 36.66±9.12 with a majority (70.72%) exhibiting a positive attitude towards genetic testing. Factors like age, job, and marital status were found to be significantly associated with attitudes toward genetic testing at a 95% level of significance. Based on the study's findings, several recommendations are proposed: there should be regular training programs in order to keep the professionals updated about genetic testing. Implement continuous education programs to improve healthcare professionals' knowledge of genetics, genetic testing, and emerging technologies. Key words: Genetics, knowledge, attitude, health care professionals, genetic testing