Aim The click-speaking Khoesan of Southern Africa represent the oldest living lineage and are among the most genetically diverse, of modern human populations. In studying the extent and evolution of human immunogenetic diversity, strong balancing selection has been identified in the Khoesan HLA region. The aim of our study was to investigate the genetic and functional diversity of HLA-C and KIR interactions in the Khoesan and compare them to populations with different demographic and genetic histories. Methods We investigated the allelic diversity of KIR2DL1, 2DL2, 2DL3 (using pyrosequencing) and HLA-C (using Luminex) in 56 unrelated Khoesan hunter-gatherers from Upington, South Africa. To measure ligand-specificity and strength, KIR-Fc fusion proteins were made for each identified allotype and tested against One Lambda Labscreen HLA class I beads. To measure the population-wide combinatorial reactivity of KIR and HLA allotypes we devised a scoring system that accounted for their surface expression, binding and signaling capacity as well as their frequencies. Results We identified two novel alleles of KIR2DL1, which are common in the Khoesan. Analysis of 1000 genomes data showed these alleles are unique to this population. 2DL1 ∗ 022 encodes lysine at position 44 which changes its specificity from HLA-C2 to C1 and 2DL1 ∗ 026 binds HLA-C2 but does not transduce an inhibitory signal because it lacks a cytoplasmic tail. Together these alleles reduce the capacity of 2DL1 to function as a C2 receptor in the Khoesan. When accounting for functional characteristics of receptor/ligand pairs, the population wide reactivity of 2DL1-C2 was much lower in the Khoesan than predicted from allele frequencies alone. In contrast, the modeled reactivity of 2DL3-C1 was unaffected. We observe an inverse correlation between functionally interactive allotypes and ligand frequencies such that combinatorial reactivity is equilibrated across populations. Conclusions The Khoesan have a diverse and unique repertoire of KIR genes that have evolved under strong selective pressure from reproductive and infectious disease. Although allele frequency spectra of both loci differ significantly, the signature of these selective pressures remains in the form of KIR and HLA functional profiles that are equilibrated between human populations.