The assessment of genetic diversity and karyotypes can help to identify new species and taxonomic units and contribute to species conservation. Ecological niche modeling can be used to predict species distribution and to estimate the impact of climate changes on its survival. The Alouatta guariba group is one of the most threatened primates in the Americas, with two endemic Atlantic Forest subspecies: A. g. guariba and A. g. clamitans, referred to as Northern and Southern populations, respectively. Although extensively studied, the distribution and the genetic diversity of these subspecies remains unclear. We assessed species relationships and genetic diversity in the A. guariba group and evaluated the potential future distribution of populations. We sequenced the mitochondrial cytochrome b gene of 14 captive and 108 free-living individuals to generate a maximum likelihood phylogenetic tree and median-joining haplotype network and to estimate the nucleotide diversity between the taxa. We conducted neutrality tests to test for population expansion. We also linked ten new karyotypes, obtained from cell cultures from blood samples of captive individuals, with the positions of the clades in the gene tree. Finally, we used ecological niche modeling to predict the potential distribution of the Northern and Southern populations. We found three lineages within the A. guariba group: North A and North B associated with A. g. guariba, and South C associated with A. g. clamitans. The results showed that A. guariba group is a species complex, with reduced haplotype and nucleotide diversity within each lineage, compared with other Alouatta species. The neutrality tests and median-joining analysis suggest a recent diversification and expansion of each lineage, and the geographical data, based on the localities of the individuals that we analyzed genetically, increase the geographic distribution of A. g. guariba, expanding the distribution farther south in southeastern Brazil than previously described. Our findings show the distribution of the A. guariba group and genetic diversity of recently expanding populations, contributing to the assessment of this Critically Endangered species. We also suggest that environmental losses and climate change are major factors in the drastic population decline of the A. guariba group.