Simple Summary Phlebotomus perfiliewi is one of the main vectors of Leishmania infantum, etiologic agent of zoonotic visceral leishmaniasis (ZVL), in the Western Mediterranean basin including Tunisia. Phlebotomus perfiliewi, originally endemic only in Northern humid bio-climatic zones, became established in a highly irrigated area of the central arid bioclimatic zone. Therefore, surveillance of sandfly vectors is a cornerstone for the establishment of control strategies. An entomological survey was carried out using light traps in a humid and in a highly irrigated arid bio-climatic zone located in northern and central Tunisia, respectively. Collected sandflies were identified based on morphological criteria, and identification of Ph. perfiliewi was confirmed by molecular tools. Results indicated that Ph. perfiliewi is the most abundant sandfly species in both sites and it is genetically variable. Thus, surveillance and control of sandflies is highly needed to limit the incidence of sandfly-borne diseases. Based on nucleotide sequences, we re-identified representative samples of Phlebotomus perfiliewi originating from two different biogeographical areas of Tunisia, whose populations had previously been identified based on morphological criteria. A partial region of the mitochondrial DNA cytochrome b gene was targeted, and sandfly species was determined by analogy with DNA sequences available in the GenBank database via a BLAST analysis, taking into account the query coverage and percentage identity. The recognized species presents the most substantial homology with the analyzed sequence. The results of the molecular identification showed complete agreement with the morphological identifications, and Phlebotomus perfiliewi is genetically variable.