The invasion of non-native species, and declines or extinctions of native species, can act together to drive either biotic homogenization or differentiation between regions over time. To date, no studies have investigated whether high incidence of biological invasions, as well as high risk of extinction, among freshwater crayfishes has resulted in biotic homogenization consistent with other taxonomic groups globally. We used Jaccard’s dissimilarity index to calculate taxonomic beta diversity of native crayfishes between and within major regions of North America, the most species-rich region on the planet for these organisms, and subsequently decomposed beta diversity into its turnover and nestedness components. To estimate biotic homogenization or differentiation, we next added introduced and established non-native crayfishes to these beta diversity calculations, and then incrementally subtracted native crayfishes on a gradient of high to lower vulnerability to extinction. We found that North American native crayfishes have extremely high beta diversity between major geographic regions of this continent, driven by turnover rather than nestedness, whereas beta diversity and its components are more heterogenous within major geographic regions. Further, we found that biological invasions by introduced non-native crayfishes drive biotic homogenization, rather than differentiation, between all regions of North America, and this effect was substantially greater than that of potential future extinctions. Within major regions of North America, biological invasions resulted in either biotic homogenization or differentiation, although the magnitude of homogenization when present was greater than that of differentiation. Cumulatively, biological invasions by non-native crayfishes are driving biotic homogenization of North American crayfishes. Management interventions are urgently needed to protect native species and ecosystems from the negative effects of these invasive crayfishes, as well as to conserve the antiquity and novelty of crayfish communities across North America.