Phlebotomus chabaudi Croset, Abonnenc & Rioux, 1970 and Phlebotomus riouxi Depaquit, Killick-Kendrick & Léger, 1998 (Diptera: Psychodidae) are closely related species of phlebotomine sand flies, the females of which are suspected of transmitting Leishmania killicki Rioux, Lanotte, and Pratlong 1986 in Tunisia. Although males of these species are distinguishable by the number and size of setae on the basal lobe of the coxite, morphologically differential characters between the females are blurred. A molecular study, based on 40 Algerian and Tunisian specimens, was conducted to distinguish females of these two species. Alignment of the cytochrome c oxidase I (COI) sequences and their analysis by using neighbor-joining and maximum likelihood showed the separation of P. chabaudi and P. riouxi, irrespective of intraspecific variability. Both COI and cytochrome b have been tested as molecular identification tools. Single strand conformation polymorphism analysis did not distinguish these two species, whereas restriction fragment length polymorphism can be applied to identify P. chabaudi and P. riouxi for surveillance purposes.