Neotoma magister (Allegheny Woodrat) has a conservation status of vulnerable or at even greater risk across 13 eastern US states. A lack of recent museum vouchers at the southern limit of its distribution, where its sister species Neotoma floridana (Eastern Woodrat) occurs, creates uncertainty as to the current range of the Allegheny Woodrat in Alabama. We opportunistically collected woodrat scat samples for species identification using mitochondrial cytochrome b comparisons that provided genetic evidence of Allegheny Woodrats in Alabama north of the Tennessee River, consistent with its recognized distribution. These new samples of Allegheny Woodrats are most closely related to Allegheny Woodrats from Pennsylvania, Virginia, and West Virginia, and with greater intraspecific cyt b variation (5%) than previously reported. Comparisons between our woodrat samples from Alabama (2 north and 2 south of the Tennessee River), and published Allegheny and Eastern Woodrat sequences revealed a clear distinction between species even at small geographic distances, with mean interspecific divergence values of over 8%. We add to the known genetic variation of Allegheny Woodrats, and further characterize their genetic differentiation from Eastern Woodrats. Additional data are needed to delimit the distributional boundaries of the 2 species in Alabama and beyond.