In a recent phylogenetic study of Mesoamerican freshwater mussels (Bivalvia: Unionidae), new molecular support for the tribe Popenaiadini was presented. Several genera of Mesoamerican mussels, including Nephronaias Crosse & Fischer, 1894, were predicted to be within the Popenaiadini tribe based on morphology, but this hypothesis had not been tested with molecular data. Several freshwater mussel specimens were recently collected in Guanacaste Province, Costa Rica. Based on the morphology of the specimens, the known distribution of Nephronaias in Costa Rica, and examination of specimens in the Zoology Museum of the University of Costa Rica, this species was identified as Nephronaias tempisquensis Pilsbry, 1920. After amplifying a gene extension characteristic of the male-transmitted genomes of freshwater-mussel species exhibiting doubly uniparental inheritance (DUI) of mitochondrial DNA, it was concluded that N. tempisquensis exhibits DUI. Phylogenetic analyses of a combination of male- and female-transmitted mtDNA gene sequences and one nuclear gene region are consistent with the hypothesis that N. tempisquensis is a member of the Popenaiadini tribe and is closely related to the Mesoamerican Psoronaias/Psorula species complex. In addition to providing novel sex-related mtDNA data, the current study presents the first DNA sequence data for the genus Nephronaias and among the first molecular data published for native freshwater mussels from Costa Rica.