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Background: Allelic variation at the FRIGIDA (FRI) locus is a major contributor to natural variation of flowering time and vernalization requirement in Arabidopsis thaliana. Dominant FRI inhibits flowering by activating the expression of the MADS box transcriptional repressor FLOWERING LOCUS C (FLC), which represses flowering prior to vernalization. Four FRI orthologues had been identified in the domesticated amphidiploid Brassica napus. Linkage and association studies had revealed that one of the FRI orthologues, BnaA3.FRI, contributes to flowering time variation and crop type differentiation. Results: Sequence analyses indicated that three out of the four BnaFRI paralogues, BnaA3.FRI, BnaA10.FRI and BnaC3.FRI, contained a large number of polymorphic sites. Haplotype analysis in a panel of 174 B. napus accessions using PCR markers showed that all the three paralogues had a biased distribution of haplotypes in winter type oilseed rape (P < 0.01). Association analysis indicated that only BnaA3.FRI contributes to flowering time variation in B. napus. In addition, transgenic functional complementation demonstrated that mutations in the coding sequence of BnaA3.FRI lead to weak alleles, and subsequently to flowering time variation. Conclusion: This study for the first time provides a molecular basis for flowering time control by BnaA3.FRI in B. napus, and will facilitate predictive oilseed rape breeding to select varieties with favorable flowering time and better adaption to latitude and seasonal shifts due to changing climate. [ABSTRACT FROM AUTHOR]