Approximately 29 species in seven genera (Chiroderma, Mesophylla, Platyrrhinus, Uroderma, Vampyressa, Vampyriscus, and Vampyrodes) compose the Subtribe Vampyressina, a group of New World leaf-nosed bats (Phyllostomidae) specialized in fruit-eating. A recent study of restriction-site variability within the mitochondrial ND3-ND4 gene region contrasts with other molecular data, including sequence data from other mitochondrial genes, by suggesting that the monotypic genus Ectophylla (E. alba) also is member of the group and is related closely to Mesophylla. In this study, we address possible explanations for why the restriction-site data appear to contradict other molecular data by performing phylogenetic analysis of DNA sequence variation (direct survey) in the ND3-ND4 region and cytochrome b gene and by re-assessing ND3-ND4 restriction-site variability in the known sequences (indirect survey). Results from analysis of sequence data reject the Ectophylla-Mesophylla hypothesis (P<0.001) and suggest four primary lineages within Vampyressina: (1) Mesophylla-Vampyressa; (2) Chiroderma-Vampyriscus; (3) Platyrrhinus-Vampyrodes; and (4) Uroderma. We also find no support for the Ectophylla-Mesophylla hypothesis in our re-analysis of ND3-ND4 restriction-site variability, and suggest the differences between molecular studies have a methodological basis.