An electrophoretic survey of 15 protein systems (22 loci) was employed to determine the genetic relationships among 9 populations (441 individuals) of South American squirrel monkeys (Saimiri sciureus sciureus, S. sciureus boliviensis, and S. sciureus ustus). Genetic markers capable of differentiating the second from the two other taxa were observed mainly in the ADA and GPI systems. Heterogeneity for ADA and CA2 between populations from opposite banks of the Jamari river was verified in S. sciureus ustus. The average heterozygosities ranged from 3% to 5%, the lowest being in S. sciureus sciureus and the highest in S. sciureus boliviensis. Low genetic distances (D = 0.001−0.057) were observed between populations within taxa or between S. sciureus sciureus and S. sciureus ustus. But both differed to a larger extent from S. sciureus boliviensis (D = 0.11 in both comparisons). There is a positive correlation between the genetic and geographic distance matrices. The three taxa are more clearly separated (D = 0.76–0.77) from the outgroup used for comparison (Cebus apella). Our data suggest that there is only one large, polytypic species of squirrel monkeys in South America, S. sciureus, forming a contiguous ring of geographical races or subspecies. Two of the most differentiated forms meet at the Peruvian Amazonia where natural hybrids and secondary intergradation have been reported. © 1993 Wiley-Liss, Inc.