Leishmaniasis is endemic in Pakistan and is wide-spread throughout the country. Polymerase chain reaction (PCR) was performed to identify the Leishmania species present in cutaneous leishmaniasis (CL) patients from new endemic areas of the central part of Sindh province, Pakistan. The PCR primers used were designed for the identification and differentiation of Leishmania (Leishmania) major and Leishmania (Leishmania) tropica species, and PCR bands at 620 and 830 bp of the parasite-specific kinetoplast DNA sequences was identified for L. (L.) major and L. (L.) tropica, respectively. Among a total of 144 DNA samples purified from the skin biopsies of clinically suspected CL patients, 108 (75%) were positive for PCR amplification. Out of the 108 cases, 105 (97.2%) were determined to be positive for L. (L.) major infection, and 3 (2.8%) were positive for L. (L.) tropica infection. It was concluded that CL caused by L. (L.) major is the main source of infection in the central part of Sindh province in Pakistan. This rapid screening technique could be used for the diagnosis of a large number of samples from skin lesions, which commonly contain other bacterial and fungal infections.