The California myotis (Myotis californicus) and the western small-footed myotis (Myotis ciliolabrum) are largely sympatric in western North America, and are especially similar morphologically such that only subtle features of their skull distinguish the two species. Previous analysis of mitochondrial DNA (mtDNA) sequence data resulted in paraphyly of these two species. Our objective was to examine genetic differences in nuclear loci between M. californicus and M. ciliolabrum, investigate their relationship with M. leibii, and to address the conflicting morphological and mtDNA data sets. We analyzed 198 amplified fragment length polymorphism (AFLP) fragments from 17 M. californicus, 16 M. ciliolabrum, and 10 M. leibii using principal coordinate (PCoA), neighbor-joining, Bayesian, and parsimony analyses. Our analyses recovered well-supported separation of M. californicus and M. ciliolabrum based on nuclear markers, suggesting the failure of the mitochondrial markers to recover monophyletic lineages was due to a lack of lineage sorting. Unexpectedly, M. ciliolabrum was paraphyletic with respect to M. leibii individuals from the eastern United States. In conclusion, our analysis of nuclear AFLP markers recovered distinct genetic lineages or clusters that corresponded to the recognized species defined by morphology, M. californicus, M. ciliolabrum, and M. leibii. We propose that these divergences are somewhat incomplete and the divergence between M. ciliolabrum and M. leibii occurred more recently than the speciation events separating the currently sympatric species M. californicus and M. ciliolabrum.