Abstract The woodrats or packrats of the genus Neotoma have been the subject of a wide array of research including paleoecology, physiology, morphological evolution, systematics, speciation, and hybridization. In recent years, much work has been done to elucidate evolutionary relationships within and between closely related species of the genus; in particular the addition of newly collected specimens from critical geographic regions has provided new opportunities for taxonomic assessment. Given these new data and their potential, parsimony (PARS), maximum likelihood (ML), and Bayesian inference (BI) analyses were conducted on DNA sequences obtained from nine individual genes (four mitochondrial loci: 12S, 16S, CoII, and Cytb; five nuclear loci: AdhI2, BfibI7, En2, Mlr, and Myh6) to estimate the phylogenetic relationships among 23 species of Neotoma. Results of these analyses depicted a wide array of phylogenetic relationships among taxa; with substantial nodal support recovered in both the ML and PARS analyses at some mid-level and terminal positions. Several individual genes, particularly 12S, AdhI2, BfibI7, CoII, and Cytb, provided support at several basal positions; however, phylogenetic resolution was limited in the other genes. A final BI analysis where the nine genes were concatenated into a single data set produced several supported clades that corresponded to previously recognized species groups (floridana, micropus, mexicana, and lepida) and the subgenus Homodontomys. Levels of genetic divergence for within-species comparisons (estimated from the Cytb data set) ranged from 0.88% (N. magister) to 6.82% (N. fuscipes); for between sister species comparisons ranged from 4.68% (N. devia and N. lepida) to 12.70% (N. angustapalata and N. nelsoni); and for members within closely related clades ranged from 8.70% (N. bryanti and N. lepida) to 12.57% (N. goldmani and N. magister). Evaluations of generic, subgeneric, and species group boundaries were explored using phylogenetic principles on the DNA sequence data presented herein, as well as morphological findings from previous studies. Results obtained suggest that the most conservative taxonomic interpretation involves the abandonment of subgeneric delineations and relies on the recognition of eight species groups (cinerea, floridana, fuscipes, lepida, mexicana, micropus, phenax, and stephensi) as the backbone of the woodrat classification.