Saimiri (Cebidae) groups a complex of species and subspecies, which present a large morphological plasticity. Genetic analysis is complicated by the absence of consensus on classification criteria and the paucity of molecular tools available for the genus. As the squirrel monkey is widely used in biomedical research, breeding centers have been established, but the genetic make up and diversity of many of the existing colonies is unknown precluding a rationale breeding policy. To develop a genetic typing strategy for the Saimiri breeding colony of Pasteur Institute of French Guiana, we have used Cytochrome b, a mitochondrial marker, and nuclear microsatellites. Cytochrome b sequences from wild-caught Saimiri boliviensis, Saimiri sciureus sciureus and S. s. collinsi reference specimens and captive animals identified 11 haplotypes, grouped into three distinct clades. An estimate of genetic variability within each captive morphotype, and of the extent of molecular divergence between the Bolivian, Guyanese and Brazilian breeds was obtained from the analysis of three nuclear microsatellites. Taxon-specific microsatellites enabled typing of F0-F3 animals, but did not differentiate Brazilian from Guyanese animals. Three locus microsatellite analysis of a representative sample from each generation showed no trend for loss of heterozygosity, and identified hybrid animals between Bolivian and the two others sub-species. These data provide novel evidence for taxonomic classification and a rationale strategy to further type the whole colony.