Little is known about genetic variation in the 6–8 subspecies of Colobus angolensis, currently distinguished by pelage differences. We present a comparative genetic analysis of one of these subspecies, C. a. palliatus, in Kenya and Tanzania that assesses evolutionary relationships and patterns of mitochondrial genetic diversity in 103 individuals across its geographic range. Fecal samples from approximately 156 individuals were collected in four localities: (1) Diani Forest, Kenya; (2) Shimoni, Kenya; (3) Udzungwa Mountains National Park, Eastern Arc Mountains, Tanzania; and (4) Mount Rungwe, Southern Highlands, Tanzania. These samples represent at least six groups, with 5–15 samples from each. Comparative sequence analysis of a 1,795 base pair mtDNA fragment revealed 19 unique haplotypes in four populations. Phylogenetic analyses suggest that sampled Kenyan haplotypes are paraphyletic, with one Kenyan haplotype basal to all other sampled haplotypes. Analysis of molecular variance (AMOVA) suggests high levels of genetic variation among populations (ΦST 0.72, P<0.001). Genetic data are concordant with a subspecies level differentiation between C. a. palliatus populations in Kenya and those in Central and southern Tanzania, as earlier suggested based on pelage differences. This study highlights the evolutionary distinctiveness of Kenyan populations of C. a. palliatus relative to Tanzanian populations. Although C. a. palliatus habitat in Tanzania is currently better protected than in Kenya, our results suggest Kenyan and Tanzanian populations should be considered distinct units, and the protection of C. a. palliatus habitat in Kenya, as well as habitat connectivity between Kenyan populations, should be prioritized for conservation and management. Am. J. Primatol. 72:715–724, 2010. © 2010 Wiley-Liss, Inc.