AbstractSouth Africa has a large captive lion (Panthera leo) sector, but detailed knowledge on the origin of individuals and any potential genetic value to conservation targets is lacking. In 2021, the South African government committed to closing the sector and have since appointed a Ministerial Lion Task Team (2022) to initiate this process. Some have suggested that captive lions could be integrated into wild populations as part of the process but information on the genetic origins and diversity of captive lions is critical if this is to be explored further. Both the Biodiversity Management Plan for lions in South Africa (2015) and a High-Level Ministerial report for the South African government (2021) have called for more information on the genetic composition of captive lions. To determine the probable origin of captive lions in South Africa we summarised existing survey responses from captive facilities (collected 2017–2018) and CITES permit data (issued 1991–2019). Survey data suggest that most lions were sourced from within the South African captive sector. However, many CITES permits were also issued for the import of lions from across Africa and beyond, indicating possible mixed origins within the sector. To evaluate genetic relationships between captive and wild lions in South Africa we standardised existing microsatellite marker data from three laboratories and analysed genotypes of captive lions from 31 properties. A comparison of captive and wild lion genotypes revealed that the genetic composition of captive lions is currently comparable to existing wild South African lions. Captive lions cluster with similar probabilities to three of four regional reference populations of wild lions included in the study and no major signatures of inbreeding were identified. However, captive lions are highly genetically interconnected across properties and represent a smaller effective population size compared to Kruger National Park, the largest population of wild lions in South Africa, suggesting some risk of future inbreeding. There were also signatures of genetic drift which should be investigated further as it will likely compromise any potential conservation genetic value of captive lions in the future. The findings of this study should be considered when planning the fate of individuals within South Africa’s captive lion sector and within the broader context of African lion conservation.