Simple sequence repeat (SSR) markers were used to characterize genetic diversity in 30 sorghum [Sorghum bicolor (L.) Moench] accessions conserved in the national gene bank of Botswana. This collection represents the three sorghum-growing agro-ecological zones in the country. Ten microsatellite primer pairs were used, and these generated a total of 53 alleles with three to seventeen alleles per locus across the 30 accessions. A high level of genetic variation was observed among the accessions (70% of the variation), and 30% of the total genetic variation was observed within accessions. The analysis of molecular variance on sorghum accessions grouped based on agro-ecological region, ethnicity, and sorghum race revealed a non-significant variation, indicating that the alleles are distributed all over the country. Cluster analysis also did not group the accessions according to agro-ecological region, race or ethnicity. The results of this study have indicated that substantial genetic diversity exists among sorghum accessions conserved in Botswana, but that agro-ecological region, ethnicity and race did not influence the distribution of this diversity.