Although widely detected in marine ecosystems, microplastic pollution has only recently been documented in freshwater environments, almost exclusively in surface waters. Here, we report microplastics (polyethylene microbeads, 0.40–2.16 mm diameter) in the sediments of the St. Lawrence River. We sampled 10 freshwater sites along a 320 km section from Lake St. Francis to Québec City by passing sediment collected from a benthic grab through a 500 μm sieve. Microbeads were discovered throughout this section, and their abundances varied by four orders of magnitude across sites. Median and mean (±1 SE) densities across sites were 52 microbeads·m−2 and 13 832 (±13 677) microbeads·m−2, respectively. The highest site density was 1.4 × 105 microbeads·m−2 (or 103 microbeads·L−1), which is similar in magnitude to microplastic concentrations found in the world’s most contaminated marine sediments. Mean diameter of microbeads was smaller at sites receiving municipal or industrial effluent (0.70 ± 0.01 mm) than at non-effluent sites (0.98 ± 0.01 mm), perhaps suggesting differential origins. Given the prevalence and locally high densities of microplastics in St. Lawrence River sediments, their ingestion by benthivorous fishes and macroinvertebrates warrants investigation.