As historically recognized, three stygobitic species of the subgenus Aviticambarus, genus Cambarus inhabit Mississippian limestone caves along the southern edge of the Southern Appalachians and Highland Rim in southeastern Tennessee and Northern Alabama, U.S.A. These include Cambarus hamulatus, C. jonesi, and C. veitchorum. All stygobitic members of the genus Procambarus inhabit caves in Florida, Cuba, and Mexico with exception of P. pecki (in the monotypic subgenus Remoticambarus), which exists in only three caves with C. jonesi in Northwestern Alabama. It was hypothesized that Procambarus pecki was derived from a primitive Procambarus stock that gave rise to the genera Cambarus and Orconectes based on the morphological shapes of the gonopods. Excluding the unsampled rare C. veitchorum, here we present 16S rDNA phylogenetic evidence, contrary to former morphological-based inferences, for the recognition of five distinct Aviticambarus lineages including P. pecki. Cambarus laconensis is a new species restricted to one locality in Northern Alabama along the southern border of the Highland Rim. Cambarus speleocoopi is also a new species of subterranean crayfish restricted to Marshall County, Alabama. These two cryptic species, with distributions that do not overlap any other stygobitic species, were discovered during a previous phylogeographic survey of cave crayfishes in the Southern Appalachians. For cave crayfishes in particular, similar morphology owing to convergent evolution in replicate subterranean environments, obscures phylogenetic relationships and cryptic stygobitic lineages.