Two PCR methods were compared for their sensitivity in detecting cultured Leishmania major, before being used to estimate infection rates in female sandflies (Phlebotomus papatasi) collected from peridomestic animal shelters and the nearby burrows of the gerbil reservoir hosts, Rhombomys opimus, in Isfahan province, central Iran. A semi-nested PCR was used to amplify a fragment of minicircle kinetoplast (k) DNA with a length and sequence diagnostic for L. major, and a nested PCR was developed to amplify a fragment containing the internal transcribed spacers of the ribosomal RNA genes (ITS-rDNA) with a sequence diagnostic for L. major. The semi-nested PCR was less sensitive than the nested PCR when using DNA extracted from cultured promastigotes of L. major, but it was more sensitive for detecting L. major in wild-caught sandflies. At the edges of two Isfahan villages, infection rates were significantly higher in P.papatasi collected outside gerbil burrows (14/28) compared with those from peridomestic animal shelters (2/21). This is the first record of L. major detected in P.papatasi from peridomestic sites in Isfahan province.