Pearl millet (Pennisetum glaucum L.) is an important staple food crop, especially in arid and semi-arid regions of West Africa. However, the genetic diversity of pearl millet in these regions is poorly understood, and yet it is useful in identification of landmarks for in situ germplasm conservation and breeding programs, particularly in establishment of heterotic groups. Therefore, the aim of this study was to assess the patterns of genetic diversity and relationships among 30 West African pearl millet accessions, using 21 microsatellite markers. Population structure and population genetic diversity were also assessed. Genotypic data was generated using the ABI Prism 3730 and alleles were called and sized using GeneMapper software version 3.7. Molecular data was analyzed using DARwin 4.0, PowerMarker 3.0 and Arlequin version 3.11. The average marker quality index was 0.29 while the total number of alleles detected was 171 with an average of 8 alleles per locus. The average polymorphic information content (PIC) was 0.64. The average Nei's genetic diversity and heterozygosity were 0.533 and 0.433, respectively. Hierarchical partitioning of genetic variation by analysis of molecular variance (AMOVA) demonstrated that genetic differentiation was low but significant at p<0.003 with a fixation index (Fst) of 0.154. Of the total genetic diversity, 5.8, 9.5 and 84.6% were attributed to country differences, population differences within the countries and differences within populations, respectively. Cluster and principal coordinate analysis of the 30 populations revealed two distinct pearl millet accessions while the 49 individuals revealed four clusters, all on the basis of geographic origins and genotypes. This study demonstrates the utility of simple sequence repeats (SSR) markers in detecting polymorphism, estimating genetic diversity and assigning heterotic groups in a highly crosspollinated species such as pearl millet.