The Karretjie people of the South African Great Karoo are itinerants who subsist by sheep shearing. Although officially classified 'Coloured', they are aware of their Khoe and San roots. To investigate the maternal and paternal ancestries of the Karretjie people we analyzed their mitochondrial and Y-chromosome DNA variation. Their genetic ancestry was compared with a neighboring group of 'Coloured' individuals. We found that the mitochondrial DNA (mtDNA) haplogroup L0d was present in all the Karretjie people examined, suggesting a maternal contribution, exclusively from the Khoe and San, whereas the paternal ancestry in males was more heterogeneous. The Coloured sample, on the other hand, were found to have a lower frequency of L0d (64.5%), but did harbor other African (27.6%) and non-African (7.9%) mtDNA haplogroups. Similar to the Karretjie people, the Y-chromosome lineages identified in the Coloured group had heterogeneous origins. This study also enabled an assessment of mtDNA variation within L0d sub-haplogroups. All seven of the L0d sub-clades were identified in the combined sample and were used to construct an L0d network.