Background Visceral leishmaniasis (VL) is a major public health problem in Bangladesh with the highest disease burden in the Mymensingh District. The disease is transmitted by sand fly bites, but it may also be transmitted through blood transfusions. No information is available about the prevalence of Leishmania infection among blood donors in Bangladesh; therefore we aimed to investigate this question. Methods The study was carried out in the Blood Transfusion Department of Mymensingh Medical College Hospital. One thousand one hundred and ninety five adult healthy blood donors attending in this department were enrolled in the study from August 2010 to April 2011. After obtaining written consent, socio-demographic data and a detailed health history were collected. The medical officer in the unit performed a complete physical examination to exclude any acute or chronic diseases, which was followed by sero-diagnosis for exposure to Leishmania by rK39 strip test using finger prick blood. Blood donors with a positive rK39 strip test underwent a PCR test for detection of leishmania DNA in their peripheral blood buffy coat. Results Eighty two percent of enrolled blood donors were male (n=985) and 18% (n=210) were female. The mean age of blood donors was 27 years (SD, 7.95 years). The majority of donors were literate and had mid-to-higher socioeconomic condition reflected by household conditions reported by the subject. Only 2.6% had a family member with VL in the past. Three blood donors were positive for leishmania infection by rK39 strip test (0.3%, 95%CI, 0.05%-0.73%). None of these 3 had active leishmania infection as demonstrated by PCR analysis. During six months of follow up, neither rK39 positive (n=3) nor rK39 negative (n=1192) donors developed VL. Conclusion The prevalence of Leishmania donovani infection among blood donors attending the Blood Transfusion Department of Mymensingh Medical College Hospital was very low. Therefore the chance for transmission of VL through blood transfusion is negligible. We believe that the National VL Elimination Program does not need set up routine screening for Leishmania donovani infection in blood transfusion departments located in VL endemic areas of Bangladesh.