Phylogenetic relationships among all 18 species of the genus Glycine were inferred from nucleotide sequence variation in the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Pairwise sequence divergence values ranged from 0.2% (a single nucleotide) between Glycine max and Glycine soja to 8.6% between Glycine hirticaulis and Glycine falcata. The length of the ITS1 and ITS2 sequences ranged from 215 to 238 nucleotides and from 205 to 222 nucleotides, respectively, and that of 5.8S was 168 nucleotides across all the species. Phylogenetic analyses of the ITS region clearly resolved all the genomic groups that were established previously based on cytogenetic and biochemical studies. Based on this study, we assign new genome symbols: HH to Glycine arenaria, H1H1 to Glycine hirticaulis, H2H2 to Glycine pindanica, II to Glycine albicans, and I1I1 to Glycine lactovirens. Parsimony analysis of the entire ITS region, using subgenus Soja as outgroup, resulted in a trichotomy consisting of the clades: G. falcata (F genome), Glycine cyrtoloba and Glycine curvata (C genome), and all other species (A, B, D, E, H, and I genomes) of the subgenus Glycine.