Sand flies were collected from 55 locations in Jordan covering all governorates during May, 2019–October, 2019. A total of 595 sand fly specimens were collected and were identified. Eight species of genus Phlebotomus: Ph. alexandri, Ph. tobbi, Ph. perfiliewi, Ph. major syriacus, Ph. jacusieli, Ph. papatasi, Ph. sergenti, and Ph. canaaniticus were recorded in this study. DNA barcoding and molecular characterization for collected Phlebotomus sand fly species were performed. Molecular work confirmed the morphological identification. This study shows the presence of low genetic differentiation within Ph. papatasi, Ph. alexandri, and Ph. sergenti populations which could be due to high gene flow, suggesting that gene flow is not restricted by geographic distance or climate for our samples.