The high biological diversity of tropical forests has been attributed to various biogeographic mechanisms promoting diversification. Among these, the riverine barrier hypothesis postulates that populations of a species that become isolated on opposite sites of a major river may gradually diverge to form separate lineages. Brown spider monkeys (Ateles hybridus) are Critically Endangered primates are currently distributed along both banks of the Magdalena River in Colombia. Based on their pelage coloration, populations of A. hybridus on opposite sides of the river have been proposed to belong to two different subspecies: A. h. brunneus on the west bank and A. h. hybridus on the east bank. We sequenced portions of the noncoding HVI region of the mitochondrial D-loop (N = 41) and the COII gene (N = 35) from a total of 51 individuals from populations along both banks of the Magdalena River with the goal of evaluating the role of the river as a barrier to gene flow in this endangered primate. Mitochondrial DNA haplotypes were shared between populations on both banks and we found no evidence of highly structured populations occupying opposite banks of the river, suggesting that the Magdalena River has not acted as an insurmountable barrier for brown spider monkeys. Population genetic analyses also reveal likely gene flow between banks, and only a minor portion of the genetic variation currently found in brown spider monkeys can be explained by the river acting as a barrier or by isolation by distance. Our study provides evidence suggesting that the Magdalena River has not acted as a major barrier influencing the evolution of brown spider monkeys and suggests that the subspecific taxonomy of one of the most endangered primates in the world may need reexamination.