Objective: To study Leishmania infection in cats and its potential role in transmission of the disease to human by parasitological, serological and molecular methods in Ahar District, East Azerbaijan Province. Methods: In this study, 65 cats from different parts of Ahar Province were trapped. The cats were anesthetized with chloroform and blood samples were taken from jugular vein and tested by direct agglutination test. Spleen and liver smear samples were prepared in order to microscopically examine these organs, and also cultured in Novy-MacNeal-Nicolle and Roswell Park Memorial Institute 1 640 media. Finally, spleen tissue DNA was extracted to perform polymerase chain reaction analysis. Results: In direct agglutination test, 4 (6%) cats had a positive titer, while 14 (22%) cats had a titer of 1:80 which was suspected for an infection and 47 (72%) cats were negative. Culture results were negative and in polymerase chain reaction no amplification was observed. Conclusions: We found no case of feline visceral leishmaniasis. It needs more extensive studies by using a larger number of cats to firmly establish leishmaniasis in this area.