The Brassica genus comprises various important species, of which three diploid species, B. rapa (A genome), B. nigra (B), and B. oleracea (C), yielded three different pair-wise amphidiploids: B. juncea (AB), B. napus (AC), and B. carinata (BC), showing the "triangle of U". Although DNA sequences of many genes have been analyzed to reveal the relationships between A, B, and C genomes, the phylogeny of any single-copy nuclear gene has not supported the entire relationships of U's triangle. Most nuclear genomic sequences of plants have genetically recombined between alleles in inter-specific hybrids, while we recently found that intron 19 and nucleotide tag (Ntag) sequences of the single-copy nuclear PolA1 gene, encoding RNA polymerase I's largest subunit, had rarely recombined during the introgressive hybridizations in Aegilops speltoides. Because phylogenetic analysis including recombined sequences cannot reveal the phylogeny before the recombination occurred, only analysis of non-recombinational DNA sequences can resolve the true evolutionary route. In this study, the phylogenetic relationships of the PolA1 gene in the six Brassica species were clearly consistent with U's triangle. In addition, two groups of B. napus were shown divergently to have originated from the amphidiploidization between B. oleracea and two progenitors of B. rapa.