The Neotropical genus Gardnerycteris has been recently erected and described as a result of a morphologically-based phylogenetic analysis of Phyllostominae with emphasis on representatives of Mimon sensu lato. As currently understood Gardnerycteris is formed by two species, G. crenulatum and G. koepckeae. However, it has been hypothesized that G. crenulatum might be a complex of species, from which the form G. c. keenani exhibits the clearest morphological differences from other populations of ‘crenulatum’. Species limits and relationships among forms of Gardnerycteris have been only partially explored on the basis of morphological variation. Herein, we obtained partial sequences of two mitochondrial genes (cytb and COX1) and one nuclear gene (RAG2) from representatives of G. koepckeae and G. c. keenani in order to clarify the relationships within the genus. Furthermore, we performed qualitative and quantitative morphological comparisons among G. crenulatum sensu stricto, G. c. keenani, and G. koepckeae to limit species. Our results support the monophyly of Gardnerycteris, as well as the molecular and morphological differentiation of G. c. keenani from other congeners. In the light of the results, we propose changes in the taxonomy of the genus, raising ‘keenani’ to the species level.