The establishment of heterotic groups and heterotic patterns is crucial to a successful maize hybrid breeding programme. Molecular markers can be used for differentiating maize into heterotic groups which can be used for maximum exploitation of heterosis. A core set of 45 maize inbred lines was selected from 96 maize inbred lines that were obtained from major breeding programmes in Zambia, Zimbabwe, CIMMYT, IITA, and USA. The 45 inbred lines were assessed for their genetic diversity and assigned to different heterotic groups using 129SNPs. The genetic distance ranged from 0.03 to 0.99, with the highest distance observed between inbred lines B73 and Mo17 and the least between L3233 and N3. The inbred lines were clustered into four groups which corresponded to the N, SC, BSS, and Lancaster heterotic groups. The genetic divergence among temperate inbred lines was larger than that among tropical inbred lines. Temperate inbred lines with potential to improve the heterotic response of the N and SC heterotic groups were identified. The study has shown that including temperate inbred pairs from established and well-known heterotic groups is recommended for effective molecular characterisation of Southern African maize inbred lines. It is recommended that the genetic distance based grouping should be verified by combining ability studies.