The level of genetic diversity among the available breeding materials determines the potential success of a breeding program. In this study, 92 maize inbred lines were genotyped with 3047 single-nucleotide polymorphism (SNP) markers using a Kompetitive allele-specific polymerase chain reaction (KASP^TM) genotyping protocol. The objectives were to determine the level and pattern of genetic diversity and define potential heterotic groups of maize inbred lines developed by the Agricultural Research Council maize program of South Africa. More than 91% of the SNPs used were polymorphic with a mean polymorphic information content of 0.36. Gene diversity ranged from 0.35 to 0.37, with a mean of 0.36. Cluster analysis revealed the presence of three distinct subpopulations. Analysis of molecular variance revealed low but highly significant (P < 0.0001) variations among populations and high within and among individual variations. Variation among individuals contributed 83% of the total variation, whereas variation within individuals and among populations contributed 14% and 3%, respectively. Low mean population differentiation observed in this study suggested that the inbred lines might be developed from parental genotypes with similar genetic backgrounds. The mean percentage of genetic purity among the inbred lines was 4.8, with more than 79% of the inbred lines exhibiting less than 5% heterozygosity, indicating the inbred lines are fixed. Genotypes in different clusters may be earmarked as belonging to distinct heterotic groups, and their crosses may result in better heterosis. Thus, the findings of this study set the basis for earmarking heterotic groups and parental selection.