Yellow endosperm maize can be used to reduce vitamin A deficiency among many pre-school children and women of reproductive age in sub-Saharan Africa. Assessment of the genetic diversity of tropical yellow endosperm maize inbred lines will have genetic gains in breeding design to develop lines with an enhanced level of provitamin A. We screened 122 tropical yellow endosperm maize inbred lines with 62 simple sequence repeat (SSR) markers and 51 SSR loci were polymorphic. We detected 190 alleles with an average of 3.72 alleles per locus, and polymorphic information content values among the inbred lines varied from 0.12 to 0.74 with an average of 0.43. Genetic distance (GD) values among all the pairs of the 122 inbred lines varied from 0.02 to 0.61 with an average of 0.41 for the SSR markers. The inbred lines exhibited a substantial level of genetic diversity. Genetic diversity was also evaluated using carotenoid data. The Euclidean GDs varied from 1.00 to 9.97 with an average of 3.81 for the carotenoid data. Cluster and principal coordinate analyses revealed clear separation of maize inbred lines into SSR-based groupings and carotenoid-based groupings. Cluster based on SSR markers were predominantly consistent with known pedigree data of the inbred lines. The correlation using Mantel's test between carotenoid-based GD estimates and SSR marker-based GD estimates gave a low r value ( − 0.06). The grouping of lines will facilitate the selection of parental lines for making crosses to develop new lines with enhanced provitamin A content.