Background: Cutaneous Leishmaniasis (CL) is endemic in many parts of Iran. This study was conducted to investigate the fauna and some biologic factors of sand flies and detect CL vector(s) in Kharameh district which is one of the most important foci of the disease in Fars province, southern Iran. Methods: To identify the fauna, a total of 1549 sand flies were collected from April 2014 to March 2015. To determine the monthly activity, sand flies were collected from indoor and outdoor areas of the lowland and the highland regions. Results: Ten species of phlebotomine (four Phlebotomus spp. And six Sergentomyia spp.) were identified and Phlebotomus papatasi was the dominant species (53.45%). To determine the sand flies naturally infected by Leishmania spp., 188 female sand flies (145 P. papatasi, 29 P. sergenti, and 14 P. alexandri) were subjected to polymerase chain reaction (PCR) assay. Two (13.16%) specimens of P. papatasi were found to be positive for Leishmania major. Conclusions: To the best of our knowledge, this is the first PCR detection of L. major within naturally infected P. Papatasis and fly as the main vector in this region of south Iran.