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We investigated genetic variation within and among three populations of Chersina angulata; two in the southwestern Cape (West Coast National Park and Dassen Island), of South Africa and Kleinzee in the northwestern parts of the species’ range. Mitochondrial DNA sequences (274 base pairs) of the cytocbrome bgene of 25 C. angulata identified 10 haplotypes in three lineages. Estimates of coalescence time for the three lineages ranged from approximately 1.3 to 3.8 million years, implying that the lineages’ present distribution may reflect radiation during the late Pliocene to early Pleistocene Epochs. The mtDNA sequences of Dassen island and West Coast National Park populations did not differ significantly. The geographic heterogeneity in haplotype frequency distribution suggests that the Kleinzee population represents a distinct lineage from the two southwestern populations. The observed genetic structure of C. angulata has management implications for the conservation of this species. Until more information is available, we recommend that C. angulata should not be translocated between the northwestern and southwestern regions, to prevent disruption of local gene pools.