Haplotype and gene network analyses were performed on mitochondrial cytochrome oxidase I and cytochrome b gene sequences of Lutzomyia (Lu.) ayacuchensis populations from Andean areas of Ecuador and southern Peru where the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and populations from the northern Peruvian Andes, for which transmission of Leishmania by Lu. ayacuchensis has not been reported. The haplotype analyses showed higher intrapopulation genetic divergence in northern Peruvian Andes populations and less divergence in the southern Peru and Ecuador populations, suggesting that a population bottleneck occurred in the latter populations, but not in former ones. Importantly, both haplotype and phylogenetic analyses showed that populations from Ecuador consisted of clearly distinct clusters from southern Peru, and the two populations were separated from those of northern Peru.