The role of rivers in driving molecular variation and speciation through vicariance across the Neotropics remains contested. Here, we test the river barrier hypothesis by analyzing the impact of river and mountain barriers on biogeographical distributions and molecular variation in New World monkeys (Parvorder: Platyrrhini). In this quantitative molecular assessment, we analyze variability in partial sequences of mitochondrial molecular markers (cytochrome oxidase subunit II and cytochrome b) to assess taxonomic differences and impacts of geographical barriers on molecular patterns across New World monkeys. Nearly 80% of described primate species across Central and South America are separated by geographical barriers. River width was positively associated with molecular dissimilarity on opposing riverbanks of adjacent taxa for both molecular markers. Streamflow was also positively associated, though not significant, likely due to a small sample size. Aotus and Saguinus on opposing riverbanks were far more divergent than expected based on river width, whereas Saimiri and Sapajus species were more similar than anticipated. Several presently described taxa were not supported based solely on these molecular phylogenetic markers, including Saimiri, Mico, Cebus, Sapajus, and Cherecebus. These taxonomic issues are far more common where river barriers do not exist. We found river width was significantly correlated with molecular divergence in adjacent taxa: the wider the river the greater the molecular dissimilarity for two widely used mitochondrial genes. The vast majority of species boundaries were at the interface of rivers and for those with no discernable geological barrier, we found an increased likelihood that the two presently described adjoining species were molecularly similar Some genera such as adjacent Saguinus species, had a much higher molecular similarity of adjacent species than others. Our results show both river and mountain barriers are significantly associated with reduced gene flow for the vast majority of neotropical taxa, that the geological formation of river barriers coincides with estimated speciation events, and that river width can be used as a tool to estimate molecular divergence in adjacent taxa, particularly useful in areas with limited sampling.