Seventeen Y-chromosomal short tandem repeats (YSTRs)-DYS19, DYS389I, DYS389II, DYS385a/b, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4-were analyzed in 252 unrelated male individuals from Botswana. A total of 238 unique haplotypes were identified. The discrimination capacity (DC) was 0.9444 whereas the haplotype diversity (HD) was 0.9990. A database search of the 238 unique haplotypes in the Y chromosome haplogroup database (YHRD) yielded three African American, six Sub-Saharan African, and two admixed South American matches. Five additional African-American matches were detected in the Applied Biosystems Y-STR database. RST, multi-dimensional scaling (MDS) and AMOVA were used to investigate population differentiation in Sub-Saharan Africa and in Botswana. The populations in Sub-Saharan Africa were found to be heterogeneous, with Botswana showing significant differences from its neighbors. No geographic regional or ethnic differentiation was observed within Botswana. Regional and ethnic variation can be useful in forensic working hypotheses.