Accurate phylogenies are important for understanding the evolutionary histories of organisms, their reproductive traits and ecological habits. The freshwater mussel order Unionida is currently thought to include six families. However, assignment of particular species to these families has been unstable, particularly for species that have been described solely on conchological characters. Unio polystictus Heude, 1877 represents such a species. Based on DNA sequence data from five genes (COI, 16S rRNA, 18S rRNA, 28S rRNA and histone H3) and complete mitochondrial genomes, we investigated the phylogenetic position and generic affinities of U. polystictus using various analytical methods. Both the five-gene and mitogenome datasets strongly supported transferring U. polystictus from Margaritiferidae to Unionidae as Aculamprotula polysticta, comb. res. Our results also supported the following intrageneric relationships: (Aculamprotula tortuosa, ((Aculamprotula polysticta, Aculamprotula scripta), (Aculamprotula fibrosa, Aculamprotula tientsinersis))). In addition, by comparing the morphological features of Aculamprotula (Unionidae, Unioninae), Lamprotula (Unionidae, Gonideinae) and Gibbosula (Margaritiferidae, Gibbosulinae) species, potential issues of relying solely on shell morphology for high-level classification of freshwater mussels are highlighted. Confirmation of classification position and genetic relationship for Aculamprotula polysticta will helpful to understand the ecological characteristics, reproductive strategies and host-fish requirements, which can be inferred from closely related taxa.