BACKGROUND: Ethiopia is one of the top 10 countries in the world where 90% visceral leishmaniasis cases are reported. Metema-Humera lowlands are the most important foci in Ethiopia. Blood transfusion in visceral leishmaniasis endemic foci in Ethiopia does not consider screening of visceral leishmaniasis during blood donation. The aim of this study is therefore, was to assess asymptomatic Leishmania donovani infection and associated factors among blood donors attending at Metema district Blood Bank, Northwest Ethiopia. METHODS: A Health facility based cross-sectional study was conducted at Metema Blood Bank from February to March 2020. A total of 205 blood donors were eligible and participated in this study. Structured questionnaire were used to collect data on socio-demographic characteristics and perceived risk factors associated with asymptomatic visceral leishmaniasis among blood donors. Blood donors were screened using both rK39 and direct agglutination tests based on the manufactures' instructions. Data were analyzed using SPSS version 20.0. Chi-square test was used to assess associations of Leishmania donovani infection with predisposing factors. Associations were considered statstically significant on P-value < 0.05 at 95% confidence level. RESULTS: Of the total 205 participants, 32(15.6%) were positive for asymptomatic Leishmania donovani infection at least by one of the diagnostic tests used. Eight (3.9%) and 30(14.6%) of the participants` were positive by the rK39 and direct agglutination tests, respectively. Six (2.9%) donors were tested positive by both diagnostic tests. Family history of visceral leishmaniasis (X²=11.334, P = 0.003) and having neighbors with history of visceral leishmaniasis (X²=5.923, P = 0.015) were significantly associated with asymptomatic Leishmania donovani infection among blood donors. CONCLUSIONS: The prevalence of asymptomatic Leishmania donovani infection was 15.6%. Asymptomatic visceral leishmaniasis was significantly associated with donors' family and neighbors' history of infection. Therefore, laboratory screening of blood donors for visceral leishmaniasis in endemic areas will be mandatory. Moreover, this study will give base line information for future study in the country.