Visceral leishmaniasis (VL) is a serious health problem in Iran. The disease is mainly transmitted by sand fly bites, but its transmission through transfusion in endemic areas may also occur. The current study aimed to determine the prevalence of Leishmania infection among blood donors in VL-endemic areas in south of Iran. A total of 2003 healthy blood donors from blood service centers in five VL-endemic districts in Fars province, southern Iran, were enrolled in the study. The blood samples were assessed for antibodies against Leishmania infantum by direct agglutination test (DAT). Seropositive subjects were tested for the presence of L. infantum DNA in their buffy coat by the molecular method. Socio-demographic features of the subjects were also documented during sample collecting. The mean age of participants was 36.3 (SD = 10.7 years). Male constituted 94.7 % of the subjects while only 5.3 % of donors were female. Twenty-eight blood donors (1.4 %) were positive for Leishmania infection by DAT. Only one of these seropositive donors was positive for Leishmania infection by polymerase chain reaction. A significant correlation was found between age, the place of residence and seropositivity to Leishmania (P < 0.05). Findings of this study revealed that the prevalence of Leishmania infection among blood donors in transfusion centers in the VL-endemic areas in Iran is relatively high. These asymptomatic blood donors may constitute a risk of transmitting of VL to susceptible recipients.