Leishmaniasis is a serious health problem in various endemic areas. There are reports that the parasite can be transmitted via blood transfusions. We studied the clinical utility of flow cytometry for the screening of blood donors in an endemic area in Greece. Samples from 2000 blood donors from the area of Lasithi, Crete, Greece were examined by flow cytometry after labelling with a polyclonal anti-leishmania antibody conjugated with fluorescein-isothiocyanate derived from infected canines in the area. The same blood samples were simultaneously examined for serum anti-leishmania antibodies, May-Grünwald staining of peripheral blood smears and polymerase chain reaction (PCR) in buffy coat to the minicircle of kinetoplastic DNA. Direct sequencing of the PCR-amplified area helped discriminate leishmania species. Flow cytometry detected 33 cases with parasites in the peripheral blood leucocytes (1.65%), which were confirmed by PCR. One PCR-positive case was negative by flow cytometry. After prestorage leucodepletion, no sample was positive by PCR. Anti-leishmania antibodies were positive in 304 (15%) cases. Flow cytometry was found to be a sensitive and rapid method of detecting leishmania in peripheral blood samples. Leucodepletion effectively reduces the detection of the parasite, thus minimizing the potential risk of leishmania transmission through blood transfusions in endemic areas.