Aquatic communities are highly threatened by anthropogenic and climate change. However, despite their importance in these communities, information regarding temporal changes in populations and assemblages of North American crayfish is scarce. Long-term monitoring of crayfish populations in south-central Ontario, Canada, indicates that the populations are in a significant state of decline. We sought to determine whether these population declines are spatially and taxonomically broad, and if so, what factors might be associated with the declines. We sampled crayfish abundance (catch per unit effort) in 100 lakes, and compared current abundances to survey results from the early 1990s. Abundances of all species (natives and nonnatives) declined significantly during this interval. Declines were both severe (63–96% loss of abundance) and geographically widespread for all species. Previous studies have documented native species declines caused by the invasive crayfish Orconectes rusticus, but this species was absent from almost all lakes and was not a factor in the declines. We hypothesize that the introduction of predatory smallmouth bass (Micropterus dolomieui), increases in Al concentrations, and reduced Ca concentrations in these lakes are negatively affecting crayfish populations.