The giraffe (Giraffa camelopardalis) is distributed throughout sub-Sahara in savannah habitat. It is currently listed as Vulnerable on the IUCN Red Data List, as their numbers are declining. Little is known about the genetic characteristics of giraffe in South Africa. This molecular analysis of the introduced giraffe populations in the Free State Province thus provides new insights into the species’ population genetics across the Province. The specific aims of this study were to quantify the levels of genetic diversity within individual giraffe populations; and to determine the genetic structure of Giraffa camelopardalis in the Free State Province. For this purpose, a total of 129 faecal samples were taken from 20 populations within the Free State Province, and one population from the Northern Cape Province; and with reference sequences from all currently recognized sub-species taken from GenBank. Genetic diversity and genetic differentiation was quantified using sequence data from the Cyt b and D-loop mtDNA regions. Two haplotypes were identified for the Cyt b gene region, with 10 haplotypes identified for the D-loop region. Nucleotide diversity ranged from 0 to 0.132%. The results obtained indicated low levels of genetic diversity within isolated populations; however, there was more diversity present in the larger populations in comparison to the smaller populations, and even higher levels within pooled populations that can potentially be managed as a metapopulation. Various approaches to reconstruct relationships among populations, including Maximum Likelihood, a Bayesian approach and haplotype networks, showed very similar results. The results portrayed northern and southern groups when all samples and reference material were included, with individuals from the current study clustering with the southern clade. Population pairwise FST values and other measures of differentiation confirmed the strength. The extralimital giraffe population in Central South Arica was thus found to consist of more than one subspecies, with G. c. angolensis (or possibly G. c. giraffa x G. c. angolensis hybrids) surprisingly detected in a number of populations. Several recommendations were formulated in terms of the future management and conservation of giraffe in Nature Reserves and private game farms in the Free State Province. The most practical approach for dealing with inbreeding would evidently be to exchange individuals between populations, but this should be supplemented by measures such as the implementation of a database for the Province and monitoring. A metapopulation approach to conserving genetic diversity is strongly recommended, since giraffe frequently occur in low numbers and this situation is unlikely to change. To enhance future studies, sequences of nuclear genes, as well as microsatellite markers should be added to supplement the current mtDNA-based data. Improved geographic coverage within South Africa, and specifically including naturally-occurring populations, would also be beneficial.