Immunological, parasitological, and molecular techniques were applied to blood samples of dogs to diagnose Leishmania infections. In 1997, 644 domestic dogs were studied. Peripheral blood samples were collected for serological diagnosis and detection of Leishmania parasite by polymerase chain reaction (PCR). The indirect immunofluorescence test was positive in 139 (21.6%) of 644 dogs examined. The PCR was performed in 70 blood samples and 3 bone marrow aspirates. A 120-bp fragment specific for Leishmania was present in PCR hybridization analysis of all seropositive samples in the molecular assays. The PCR hybridization test, which used a minicircle of Leishmania chagasi as a probe, was negative in 20 seronegative dogs. These results suggest that a combined PCR-Southern hybridization technique is a highly sensitive approach to diagnose leishmaniasis in dogs, which are a zoonotic reservoir of leishmaniasis for humans.