The current unprecedented rates of environmental perturbation, particularly in rain forest ecosystems, are jeopardizing the persistence of a variety of tropical species.The development of adequate conservation programs requires incorporating the evolutionary history and population genetic information of species, especially in those threatened by habitat loss and fragmentation. Mexican mantled howler monkeys (Alouatta palliata mexicana) represent the northernmost distribution of primates in America, a Critically Endangered species mainly inhabiting forest remnants. We assessed historical and contemporary patterns of genetic variation in A. p. mexicana populations from five regions across its geographic distribution in Mexico. We employed non-invasive sampling techniques and evaluated microsatellite loci and mitochondrial cytochrome b sequences from 127 individuals from 15 wild and two semi-captive populations. Our data demonstrate negative genetic effects on A. p. mexicana as a result of isolation, fragmentation, and small effective population size.Results revealed two mitochondrial lineages and three genetically differentiated nuclear clusters, along with reduced nuclear genetic diversity, limited gene flow, and significant inbreeding, associated with concurrent processes of historical dispersion and contemporary landscape changes. Accordingly, we argue that A. palliata mexicana in Mexico is an independently evolving unit that meets the criteria for being assigned as an Evolutionarily Significant Unit, crucial for the preservation of the howler monkeys' phylogenetic and functional diversity. The three genetic clusters identified are essential for the maintenance of the adaptive diversity and long-term survival of this howler subspecies. Our genetic approach and conservation recommendations may be useful for other endangered primates inhabiting fragmented populations.