The production of African nightshade is below its potential due to a lack of superior varieties. The development of varieties through breeding programmes has been initiated in order to supply farmers with quality seeds. However, systematic studies on the agronomic performance of African nightshade germplasm have been started only recently, and publications in this area are very scarce. In this study, we analysed morphological traits relevant for the agronomic performance of 54 entries comprising two species: Solanum villosum and S. scabrum . In addition, AFLP markers and newly developed SSR markers were used to assess genetic diversity and differentiation in the African nightshade entries. An analysis of molecular variance showed higher variation within than among entries. A cluster analysis grouped the entries into two clusters representing the two species. The analysis of agronomic traits revealed significant differences among entries. The genetic diversity of the released cultivars and accessions was comparable to but lower than that of the local cultivars. Additionally, the local cultivars had higher allelic richness and a larger number of unique alleles than did the developed cultivars and could serve as a useful gene pool for future breeding of superior germplasm.