Freshwater mussels have a unique life history where they rely on host fishes for the metamorphosis of their parasitic larvae (glochidia) into juveniles. To determine which host fishes may act as a potential host for freshwater mussel species, glochidia can be collected from wild-caught fish hosts and identified; however, identifying mussel glochidia to species based on morphology alone can be challenging. Genetic methods for identifying mussel glochidia may provide a more accurate approach than morphometrics. We developed a protocol for extracting, quantifying, amplifying, and visualizing glochidia DNA. Glochidia samples (10) were collected from mussels of known species (Alasmidonta varicosa and Lampsilis cariosa) from three hatcheries. Mussel tissue samples (14) were collected from six known species (A. varicosa, A. undulata, L. cariosa, L. radiata, Elliptio complanata, and Utterbackiana implicata) from the Ware River (Massachusetts, USA) and a hatchery. Using a DNA mini-prep kit, DNA was extracted from individual and pooled glochidia and tissue samples (visceral swabs). DNA was amplified via PCR using three existing primers developed for freshwater mussels and visualized using gel electrophoresis. Primers targeted the mitochondrial loci cytochrome oxidase c subunit 1 (CO1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) gene regions. Since CO1 and ND1 are highly conserved across taxa but can be variable between species, we expect at least one primer to be effective for identifying species of freshwater mussels. This protocol will be used to identify multiple species of glochidia in samples collected from wild fishes for examining the host-mussel relationship in wild populations.