We developed a multilocus deoxyribonucleic acid (DNA)-barcoding approach to identify newly transformed juvenile mussels collected from naturally infested fishes in a federally protected waterway that is home to a diverse mussel community, the St Croix River (Minnesota/Wisconsin, USA). We used new and publicly available data downloaded from GenBank to build reference databases for identified adult mussels. We assessed the efficacy of the mitochondrial loci cytochrome oxidase c subunit I (COI) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) for DNA barcoding. We concluded that the barcoding gap between average intra- and interspecific genetic distances is wider for ND1 than for COI, but both loci perform well for species identification in character-based phylogenetic analyses. Almost every species formed a monospecific clade with high bootstrap and posterior-probability support. We obtained newly transformed juvenile mussels by collecting individuals of 3 different fish species that were infested with unionid larvae. We held the fish in aquaria until the mussels emerged naturally. We then extracted DNA and sequenced our loci of interest. When sequences from the juveniles were included in phylogenetic analyses, they grouped with single species (or, in one case, a pair of closely related species) with high bootstrap and posterior-probability support. Identifying juveniles using morphology alone is difficult and, in some cases, impossible. Therefore, our approach will be useful to researchers interested in the relationship between unionid mussels and their fish hosts.