The phlebotomine sand fly, Lutzomyia verrucarum, is an important vector of cutaneous leishmaniasis in Peru. It is found associated with human habitation at elevations between 1,000-3,000 m in 3 major valleys separated by the Andean mountain ranges. Previous studies have demonstrated varying degrees of genetic isolation between valleys with the assumption that populations within valleys were panmictic. To determine the genetic structure of L. verrucarum within the valleys, 16 to 20 specimens per village and four villages per valley were used. The genetic variation was examined by amplification of fragments of the mitochondrial genome that included the 1,400 bp NADH dehydrogenase subunit 4 (ND4), 700 bp of cytochrome b (cytb) and 700 of bp cytochrome oxidase 1 (CO1). The genes were amplified and sequenced. Specimens from geographically distinct sites, Lima (coastal Peru) and Huanuco (eastern Peru) provided outgroup comparators. Cytb sequences had 118 variable sites consisting of 2% of the sequence. CO1 and ND4 sequences demonstrated a similar level of variability. The gene genealogy was visualized using the computer program TCS, gene sequences from central Peru clustered in a lineage distinct from that of Lima and Huanuco. The data indicate that genetic structuring has occurred within the 1,200 x 300 km geographic range of L. verrucarum.