Abstract Hybridization of non-native crayfishes with native crayfishes can facilitate invasion success of the non-native. We report novel genetic evidence of hybridization between rusty crayfish, Faxonius rusticus (Girard, 1852), and Allegheny crayfish, F. obscurus (Hagen, 1870), two non-native species in the upper Juniata River basin of central Pennsylvania. We used double-digest restriction-associated DNA sequencing, which yielded 28,031 single nucleotide polymorphisms, to characterize the extent of hybridization. Two of eight individuals with mixed character traits were identified as the progeny of hybrids backcrossed into rusty crayfish. One individual, presumed to be a rusty crayfish in the field, was genetically identified as a hybrid backcross to rusty crayfish. The three crayfish taxa (rusty, Allegheny, and intermediates) are virtually indistinguishable based on typical morphological characters. Key character traits that distinguish rusty and Allegheny crayfishes can be mixed atypically in certain individuals (i.e., intermediates). Our results indicate that introgressive hybridization has occurred between rusty and Allegheny crayfishes, and that at least some of the character-trait intermediate individuals have hybrid ancestry. Hybridization may be an additional means by which invasive rusty crayfish increase their fitness and dominance at the expense of existing species and their habitat.