Systematic relationships within the 5 recognized species of Glossophaga were assessed using the complete cytochrome-b gene (1,140 base pairs). Our samples from 4 species, G. commissarisi, G. leachi, G. longirostris, and G. morenoi, show little intraspecific geographic subdivision. Alternatively, G. soricina has a major mtDNA subdivision that may reflect 2 biological species. Our data suggest that G. soricina is the sister taxon to the remainder of the genus, G. leachi and G. longirostris are sister taxa, and the position of G. morenoi is unresolved, placed either with the G. leachi–G. longirostris clade or with the G. commissarisi clade. Distance values among G. morenoi, G. commissarisi, G. longirostris, and G. leachi were similar, suggesting a similar time of speciation for these 4 species.