Fourteen electrophoretically variable and 12 monomorphic erythrocytic and serum proteins were used to determine the genetic relationships among Bolivian squirrel monkeys (Saimiri boliviensis boliviensis), Peruvian squirrel monkeys (Saimiri boliviensis peruviensis), and Guyanese squirrel monkeys (Saimiri sciureus sciureus). The results supported the classification scheme of Hershkovitz (AMERICAN JOURNAL OF PRIMATOLOGY 7:155–210, 1984), which is used above. A profile of marker phenotypes can unambiguously discriminate between the two species examined, and can discriminate most S. b. boliviensis from most S. b. peruviensis. All three groups can be distinguished unambiguously by the location of the centromeres (acrocentric or submetacentric) on chromosomes 15 and 16; however, the biochemical genetic markers provide a powerful means of detecting admixture that might not be detected cytogenetically in instances where hybridization occurred earlier than the preceding generation. The use of this panel of biochemical genetic markers, combined with karyotypic analysis, can ensure a high degree of certainty that animals selected for experimental protocols are uniform with respect to unique physiological characteristics of each species and subspecies. They also can ensure that animals selected to be members of breeding colonies are of a single species/subspecies type and reproductively compatible.