About 50 species of sand flies have been reported to be prevalent in India. We explored the utility of the DNA barcode approach towards species identification of these medically important insects. A total of 62 specimens belonging to seven morphologically identified species of two genera, Phlebotomus and Sergentomyia, collected from Puducherry Union Territory, Maharashtra and Rajasthan states of India were subjected to the analysis. Neighbor-joining (NJ) analysis of DNA barcode sequences identified the individuals of seven morphological species into eight distinct species, as presented in the designed NJ tree. This methodology delineated morphologically identified species, S. bailyi, into two genetically isolated groups. Also, this study characterizes DNA barcodes of P. argentipes and P. papatasi, the vector species of leishmaniasis in India, for the first time.