Flowering time is an important adaptive trait and varies among Arabidopsis thaliana and its related species, including allopolyploids that are formed between A. thaliana and Arabidopsis arenosa. FLOWERING LOCUS C (FLC) inhibits early flowering in A. thaliana. A previous study showed that late-flowering A. arenosa contained two or more FLC alleles that were differentially expressed in Arabidopsis allotetraploids, but the genomic organization and evolution of FLC locus were unknown. Comparative sequence and evolutionary analyses were performed in FLC-containing genomic regions in A. thaliana, A. arenosa and Arabidopsis lyrata, and expression of FLC loci and alleles was examined in Arabidopsis allopolyploids. The FLC locus was tandemly duplicated in A. lyrata and triplicated in A. arenosa, and the tandem duplication event occurred after divergence from A. thaliana. Although FLC duplicates were highly conserved, their upstream sequences rapidly diverged. The third FLC copy in A. arenosa acquired a new splicing site through a point mutation in the intron and generated the new exon followed by an early stop codon, resulting in a novel MADS box gene. Flowering time variation in Arabidopsis allopolyploids is probably related to the expression diversity and/or copy number of multiple FLC loci. Moreover, exonization of intronic sequence is a mechanism for the origin of new genes.