Novel phenotypes often arise in generations immediately following polyploidization. Previous studies have shown that separate lineages derived from a resynthesized Brassica napus allopolyploid rapidly evolved heritable differences in flowering time. These early-flowering and late-flowering polyploid lines were expected to be genetically identical because they were derived from a single, chromosome-doubled amphihaploid plant. In this study, we investigated the molecular genetic basis for these flowering time differences. We assessed the diploid B. rapa and B. oleracea parents and the early- and late-flowering B. napus lineages for changes in genome structure, and for changes in transcript levels of four sets of FLOWERING LOCUS C (FLC) genes. No such changes were observed for BnFLC1, but we detected chromosomal rearrangements (e.g. de novo non-reciprocal transpositions) and changes in transcript level for BnFLC2 and BnFLC3 between the early- and late-flowering B. napus. A chromosomal rearrangement of a genomic segment containing BnFLC3 was responsible for 29% of the phenotypic variation among the B. napus lines. Expression of BnFLC5 was silenced in all polyploids, although no changes in genome structure were detected. An ongoing investigation of 50 identical B. napus allopolyploids may further reveal the dynamics of changes in phenotype, genome and transcriptome at the early stages in polyploid evolution. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82, 675–688.