Abstract: Background and Objective: Visceral leishmaniasis (VL) remains a major concern in many parts of Sudan. The disease is transmitted by Phlebotomus orientalis. The objective of this study was to determine genetic structure of Phlebotomus orientalis population from 5 geographical regions in Sudan. Materials and Methods: A total of 194 individual sand flies were collected from 5 geographic regions in Sudan. The field collected sand flies were analyzed by Random Amplified Polymorphic DNA (RAPD) using 30 primers. Results: Eight hundred and 65 bands from 4 RAPD primers were analyzed for genetic variation. A higher level of intrapopulational variability was detected in populations of P. orientalis from eastern Sudan compared to those populations from central and northern Sudan. Diagnostic bands were detected in populations of P. orientalis central Sudan. Hieratical clustering analysis showed clear clustering into 2 main populations with 1 population subdivided into 4 subpopulations. However, these populations did not show any correlation with their geographical origins. Furthermore, the low genetic differentiation among subpopulations was supported by fixation index (FST) estimated by analysis of molecular variance (AMOVA). Conclusion: It is concluded that the populations of P. orientalis from the selected areas in Sudan have a low genetic differentiation. However, assessment of genetic structure of P. orientalis populations is important for understanding the patterns of transmission of VL in different endemic areas.