This research was carried out to study the morphometric and molecular differences in populations of Phlebotomus orientalis from three different regions in Sudan. Samples of Phlebotomine sandflies were collected from Gedaref, White Nile and Khartoum States. The specimens of P. orientalis were collected using CDC miniature light traps and sticky paper traps. The sand fly specimens were then mounted on glass slide on Brelese's medium and identified to species level. Then mounted samples of P. orientalis were processed for morphometric measurements of 23 and 29 characters for females and males respectively. Furthermore Cyt b, ITS2 rDNA 18S genes of P. orientalis from different regions were amplified by conventional and Real-time PCR. Amplified samples of Cyt b and ITS2 genes were purified and sequenced. The sequences were assembled, edited, analyzed and then were blasted with the database of the GenBank. Populations of P. orientalis from three geographic areas showed some degree of differences based on both morphometric and molecular analysis.