Barnyardgrass, hexaploid Echinochloa crus-galli, is considered to arise from the hybridization between tetraploid Echinochloa oryzicola and an unknown diploid species. The genetic relationship between E. crus-galli and E. oryzicola was examined to investigate the position of E. oryzicola in the evolutionary process of E. crus-galli, based on the nuclear DNA internal transcribed spacer (ITS) and the chloroplast cpDNA trnT-L, trnL intron, and trnL-F regions. New World E. crus-galli was clearly separated from Eurasian E. crus-galli and showed a close relationship to the American taxa, Echinochloa crus-pavonis and Echinochloa walteri, in both the ITS and chloroplast DNA. The nrDNA ITS sequences indicated no differentiation between the Eurasian E. crus-galli and E. oryzicola, in contrast to their clear divergence in the cpDNA sequence. The present results suggest that E. oryzicola is the male donor of E. crus-galli.