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Sixteen RFLP loci in 62 landraces were assayed to study genetic differentiation in foxtail millet, Setaria italica (L.) P. Beauv. Among 52 bands, 47 were polymorphic among foxtail millet landraces. A dendrogram constructed based on RFLPs was divided into five major clusters (cluster I–V). Clusters I and II contained strains mainly from East Asia. Cluster III consisted of strains from subtropical and tropical regions in Asia such as Nansei Islands of Japan, Taiwan and the Philippines and India and cluster IV consisted of some strains from East Asia, a strain from Nepal and a strain from Myanmar. Cluster V contained strains from central and western regions of Eurasia such as Afghanstan, Central Asia and Europe. Chinese landraces were classified into four clusters. These results indicate that foxtail millet landraces have differentiated genetically between different regions and that Chinese landraces were highly variable.