The Appomattox River basin in Virginia is a poorly understood system for freshwater crayfish diversity and distribution. We sampled 36 locations in the upper Appomattox basin for crayfish and noted general habitat characteristics. The following 7 species of crayfish were discovered in the system belonging to 4 genera: Cambarus cf. acuminatus, Cambarus bartonii bartonii (Common Crayfish), Cambarus longulus (Atlantic Slope Crayfish), Lacunicambarus diogenes (Devil Crayfish), Procambarus acutus (White River Crayfish), Creaserinus fodiens (Digger Crayfish), and invasive Procambarus clarkii (Red Swamp Crayfish). Native C.cf. acuminatus dominates the upper Appomattox region, having a wider distribution and higher relative abundance compared to other species in the basin. All other taxa demonstrated relatively limited distributions and low abundance. The least encountered species were the 2 primary burrowing species, Devil Crayfish and Digger Crayfish, which exclusively used shallow seasonal pools during the spring breeding season. We report range extensions for C. cf. acuminatus, Atlantic Slope Crayfish, Common Crayfish, Devil Crayfish, Digger Crayfish, and Red Swamp Crayfish, and recommend additional surveys to improve our understanding of the diversity and distribution of crayfish in the Appomattox River basin. Furthermore, we recommend monitoring of the Briery Creek and Sandy River systems to assess the extent and severity of the Red Swamp Crayfish invasion. Additionally, we urge a systematic study to resolve the taxonomic uncertainty of a potentially new Creaserinus species in the Appomattox River basin.