Understanding the genetic variation among landrace collections is important for crop improvement and utilization of valuable genetic resources. The present study was carried out to analyse the genetic diversity and associated population structure of 621 foxtail millet accessions of Korean landraces using 22 EST-SSR markers. A total of 121 alleles were detected from all accessions with an average of 5.5 alleles per microsatellite locus. The average values of gene diversity, polymorphism information content, and expected heterozygosity were 0.518, 0.594, and 0.034, respectively. Following the unweighted neighbor-joining method with arithmetic mean based clustering using binary data of polymorphic markers, the genotypes were grouped into 3 clusters, and population structure analysis also separated into 3 populations. Principal coordinate analysis (PCoA) explained a variation of 13.88% and 10.99% by first and second coordinates, respectively. However, in PCoA analysis, clear population-level clusters could not be found. This pattern of distribution might be the result of gene flow via germplasm exchanges in nearby regions. The results indicate that these Korean landraces of foxtail millet exhibit a moderate level of diversity. This study demonstrated that molecular marker strategies could contribute to a better understanding of the genetic structure in foxtail millet germplasm, and provides potentially useful information for developing conservation and breeding strategies.