Leishmania (Leishmania) major has been identified as the major causative agent of cutaneous leishmaniasis in Sindh Province of southern Pakistan. To make a rational approach for understanding the pathogen transmission cycles, the sand fly species and their natural blood meals in the endemic areas were examined. Total DNA was individually extracted from sand flies collected in four villages in Sindh Province. PCR-RFLP (restriction fragment length polymorphism) and sequence analysis of the 18S ribosomal RNA gene revealed that female sand flies identified were Sergentomyia clydei/Sergentomyia ghesquierei/Sergentomyia magna (68.6%), Sergentomyia dubia (17.1%), Phlebotomus papatasi (7.4%), Phlebotomus alexandri-like sand flies (3.4%) and Sergentomyia dentata (3.4%). PCR amplification of leishmanial kinetoplast DNA did not result in positive signals, suggesting that all 175 tested female sand flies were not infected with leishmanial parasites or contained undetectable levels of leishmanial DNA. Amplification and sequencing of the vertebrate cytochrome b gene in 28 blood-fed sand flies revealed that P. papatasi fed on cattle and wild rat whereas P. alexandri-like specimens fed on human, cattle, goat and dog. Although Sergentomyia sand flies are generally known to feed on cold-blooded animals, S. clydei, S. dubia and S. ghesquierei preferred humans, cattle, goat, sheep, buffalo, dog, donkey, wild rat and Indian gerbil. The epidemiological significance of the zoophilic feeding on various host species by Phlebotomus and Sergentomyia sand flies in Pakistan is further required to study for better understanding the zoonotic transmission of sand-fly-borne pathogens and for appropriate management of the vectors.