Simple sequence repeat (SSR) molecular markers were used for genetic diversity analysis and population structure of the cultivated Pearl millet in Benin, West Africa. In order to assess the level of genetic diversity, 14 polymorphic SSR markers were used to screen 114 accessions from different agro-ecological zones in Benin. SSR markers were found to reveal a total of 57 alleles with an average of 4.071 allele per locus. Genetic diversity index varied from 0.099 to 0.633 with an average of 0.405. The average observed heterozygosity was found to reach 0.425. The analysis of molecular variance showed no real differentiation between regions. Only 5% of genetic variation was observed between samples collected from north-eastern and north-western region. A high level of variation (95%) was observed among accessions. Moreover, both principal component analysis (PCA) and the dendrogram obtained from the genetic distance among accessions revealed the absence of any specific structuration of accessions from each region under study. Our results confirmed diversity among cultivated Pearl millet in Benin and such diversity is not clustering according to geographical patterns.