The phlebotomine sand flies Lutzomyia pia (Fairchild & Hertig 1961) and Lutzomyia tihuiliensis Le Pont, Torrez-Espejo & Dujardin 1997 (Diptera: Psychodidae) belong to the pia series of the Lu. verrucarum species group, which includes several species that bite humans in Andean foci of leishmaniasis. The females of these two species exhibit isometry and isomorphism in anatomical structures of the head and terminalia commonly used in taxonomic identification of sand flies. They can only be differentiated based on subtle differences in the pigmentation of the pleura. In Lu. tihuiliensis, this is restricted to the basal portions of the katepimeron and katepisternum, whereas in Lu. pia both structures are totally pigmented. Taking into account the subtle morphological differences between these species, the objective of the current study was to evaluate the specific taxonomic status of Lu. tihuiliensis with respect to Lu. pia. A 475-bp portion of the mitochondrial genome was sequenced, composed of the 3′ end of the cytochrome b gene, intergenic spacer 1, the transfer RNA gene for serine, intergenic spacer 2, and the 3′ end of the gene NAD dehydrogenase 1. Genetic analysis confirms that Lu. tihuiliensis and Lu. pia constitute two distinct species and this is supported by four strong lines of evidence, i.e., the paired genetic distances, size differences and amino acid composition of the cytochrome b protein, presence and absence of intergenic spacer one and divergence observed in the sequence of the transfer RNA gene for serine. It also confirms the validity of the pleural pigmentation pattern as a species diagnostic character and the importance of performing a detailed examination of this character during morphological determination of phlebotomine sand flies in the series pia.