Microplastics have been identified as a widespread, persistent environmental pollutant. The investigation of microplastics in marine ecosystems has been prevalent in the literature; however, much less consideration has been given to this form of pollution in freshwater ecosystems. Relatively few studies have considered the uptake of microplastics in freshwater mussels. This study investigated the presence of microplastics in fluted-shell mussels (Lasmigona costata) collected from various sites in the Grand River watershed, Southern Ontario’s largest watershed and home to one million people. The soft tissue of adult mussels underwent enzyme digestion, followed by filtration to isolate undigested particles. Particles were removed and analyzed using Raman spectroscopy to determine their composition. Ten different polymers were identified in the sampled mussels, with polypropylene-co-polyethylene being the most prevalent. Microplastic particles were detected in 71% of mussels with the greatest number of particles observed in a single mussel being seven. No significant difference in microplastic particles per mussel was observed among the different sites sampled. A significant positive relationship between particles per mussel and size of upstream catchment was observed, but a relationship between particles per mussel and percentage of urban land use was not observed.