Ancient DNA (aDNA) analysis was employed to obtain information on the population relationships of the two Thulamela individuals (AD 1400-1700) and six other skeletons from various archaeological sites of the southern African Iron Age – Tuli (Botswana), Nwanetsi, Makgope, Happy Rest and Stayt. Although sequences were short, it seems that the Thulamela female aligns somewhat more with eastern populations as opposed to the male who aligns more with western groups. This result is not surprising given that the two individuals were buried at the same site but their burials were hundreds of years apart. It was also possible to identify genetic links between the Iron Age individuals and modern southern African populations (e.g. some of the skeletons assessed showed maternal genetic similarities to present-day Sotho/Tswana groups) and to separate the samples into at least two genetic groups. Poor quality and quantity of DNA meant that only haplogroups, not subhaplogroups, of the individuals could be traced.