Five microsatellite loci were used to develop multilocus genotypes for Neotoma macrotis (n = 128) and N. fuscipes (n = 29). Several statistical analyses were used to estimate genetic structure, levels of genetic variability, and degree of relatedness within groups of these 2 species. Samples of N. macrotis represented 2 groups and 4 population clusters throughout southern California. Samples of N. fuscipes represented 2 regions in northern and southern California. Genetic structure was detected among samples of N. macrotis and N. fuscipes at a regional level. Both species displayed moderate to high genetic diversity in terms of mean expected heterozygosity (0.939 and 0.804 for N. macrotis and N. fuscipes, respectively) and mean polymorphic information content (0.930 and 0.761 for N. macrotis and N. fuscipes, respectively). Mean relatedness values within regions and populations of N. macrotis indicated 4th-order levels of relatedness within groups (e.g., distant-cousin relationships). Mean relatedness values within regions of N. fuscipes indicated 2nd-order (e.g., half-sibling) relationships within the northern region and 3rd-order (e.g., cousin) relationships in the southern region. One locus in particular (Nma04) was determined to be diagnostic in distinguishing between these 2 species.