Sand fly-borne diseases have a significant impact on global public health, although they are typically overlooked. In the Indian subcontinent, particularly in Nepal, India, and Bangladesh, over 46 cryptic species within the genera Phlebotomus and Sergentomyia play a key role in transmitting diseases such as visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) caused by the protozoan parasite Leishmania donovani. The role of Phlebotomus (Euphlebotomus) argentipes as a primary vector for L. donovani highlights the need of accurately identifying and monitoring sand fly species in order to create efficient disease management methods. This study used both morphological and molecular techniques to examine the genetic diversity of Phlebotomine sand flies across Nepal, supporting the national VL elimination program a critical public health initiative given the disease's fatality rate, which is over 95% in untreated cases. This study assessed the DNA barcoding method's utility for identifying and measuring genetic diversity within and among Phlebotomus species collected in Nepal. A total of 315 sand fly specimens from six districts in Nepal were analyzed, together with available data from BOLD and GenBank. The identification success rate using DNA barcoding was 97%, with interspecific genetic divergence ranging from 12.23% (S. babu and S. punjabensis) to 23.45% (P. argentipes and Sergentomyia sp1). For the genus Phlebotomus , including the vector species, the overall divergence averaged 14.89%. The lowest intraspecific genetic divergence was observed in P. transcaucasicus (0.08%), and the highest in P. major (5.85%). The results confirm that the COI barcoding method is an effective tool for identifying sand fly species in Nepal, providing critical data for disease control initiatives and enhancing global understanding of vector-borne diseases.