We investigated the genetic basis for the derivation of pink coloration in petals from blue flowers in cultivated gentians. Using a revertant blue-flower phenotype that arose spontaneously from a pink-flowered cultivar, we sought to elucidate the molecular mechanism of flower color restoration caused by a suppressor mutation. Detailed sequencing analysis identified three novel deficient flavonoid 3',5'-hydroxylase (F3'5'H) alleles in pink-flowered gentians in addition to two mutations identified previously (Nakatsuka et al. in Mol Genet Genomics 275:231-241, 2006). Among the deficient alleles, one allele that contained a novel miniature inverted-repeat transposable element (GtMITE1) insertion in an intron of F3'5'H was shown to cause missplicing, resulting in abnormal F3'5'H transcripts and the pink-flower phenotype. The other two mutations were identified as a single-nucleotide insertion and gypsy-Ty3 retrotransposon (Tgt1) insertion within exon 1 and exon 2 of the F3'5'H gene, respectively. The blue-flowered revertant mutant contained a single-nucleotide spontaneous mutation immediately 3' of the TAA target site duplication and the GtMITE1 insertion, which caused restoration of normal splicing of F3'5'H and the normal blue-flower phenotype. Transient expression assays in gentian flowers in vivo demonstrated that normal F3'5'H splicing pattern was recovered from missplicing induced by the GtMITE1 insertion by the single-nucleotide substitution. These findings extend our knowledge of genomic evolution by transposable elements and spontaneous mutations in Gentiana species of economic and medical importance.