The effective use of conserved insitu and exsitu genetic resources of Bambara groundnut (BG) - an orphan crop, in pre-breeding programs is hampered by a lack of well-characterized germplasm and associated traits in the collections. Genotype-by-sequencing (GBS) is a next-generation sequencing technology that enables high-throughput SNP (single-nucleotide polymorphism) marker discovery, facilitating genomic studies and faster development of improved cultivars through genomic-assisted breeding tools. In this study, 361 accessions of Bambara groundnut from the Genetic Resources Center, International Institute of Tropical Agriculture (GRC-IITA), and individual breeding programs in Southern Africa were genotyped using single plants. We sought to determine the genetic diversity and population structure, to facilitate various downstream genomic applications of GBS-derived SNP data and to guide the formulation of a focused, trait-specific core set for Bambara groundnut. A total of 7592 SNPs were generated from the GBS pipeline. After SNP quality control filtering for missing data ≤ 15% and MAF ≤ 0.01, a total of 3848 high-quality SNPs were retained and used in the clustering analysis and investigation of population structure in the BG population. An NJ tree showed that 361 accessions were grouped into three major clusters, with two West African collections that were distinctly separated from the East and Southern African collections. The Q-matrix plot also confirmed the grouping of subpopulations into three groups; however, STRUCTURE revealed only two main groups: Southern and East Africa, and West Africa. The heatmap revealed that accessions from countries near each other were highly genetically similar, suggesting high gene flow due to seed exchanges. Due to the highly structured population by country of origin, we recommend considering proximity to capture the highest diversity.