Spider monkeys (Genus: Ateles) are a widespread Neotropical primate with a highly plastic socioecological strategy. However, the Central American species, Ateles geoffroyi, was recently re-listed as endangered due to the accelerated loss of forest across the subcontinent. There is inconsistent evidence that spider monkey populations could persist when actively protected, but their long-term viability in unprotected, human-dominated landscapes is not known. We analyzed noninvasive genetic samples from 185 individuals in 14 putative social groups on the Rivas Isthmus in southwestern Nicaragua. We found evidence of weak but significant genetic structure in the mitochondrial control region and in eight nuclear microsatellite loci plus negative spatial autocorrelation in Fst and kinship. The overall pattern suggests strong localized mating and at least historical female-biased dispersal, as is expected for this species. Heterozygosity was significantly lower than expected under random mating and lower than that found in other spider monkey populations, possibly reflecting a recent decline in genetic diversity and a threat from inbreeding. We conclude that despite a long history of human disturbance on this landscape, spider monkeys were until recently successful at maintaining gene flow. We consider the recent decline to be further indication of accelerated anthropogenic disturbance, but also of an opportunity to conserve native biodiversity. Spider monkeys are one of many wildlife species in Central America that is threatened by land cover change, and an apt example of how landscape-scale conservation planning could be used to ensure long-term persistence.