Gentians are herbaceous perennials blooming in summer through autumn. Although they are popular ornamental flowers in Japan, the regulation of their timing of flowering has not been studied. We identified and characterized gentian orthologs of the Arabidopsis FT/TFL1 gene family to elucidate the mechanisms of flowering initiation. We isolated three gentian orthologs of FT and TFL1, denoted GtFT1, GtFT2 and GtTFL1. Since up-regulation of GtFT1 and GtFT2 as well as down-regulation of GtTFL1 promoted floral initiation in gentian plantlets, these genes affected floral initiation in a similar way to Arabidopsis FT and TFL1. The expression levels of GtFT1 and GtFT2 in leaves of late-flowering gentian increased prior to floral initiation, whereas GtTFL1 was highly expressed in shoot apical meristem at the vegetative stage and decreased drastically just before flowering initiation. Comparison of gene expression patterns showed that GtFT1 expression increased earlier in early-flowering than in late-flowering gentian, whereas the timing of the increase in GtFT2 expression was similar in early- and late-flowering plants. The GtTFL1 expression in early-flowering gentian was extremely low throughout the vegetative and reproductive stages. These results indicated that either the up-regulation of GtFT1 or the down-regulation of GtTFL1 may determine flowering time. Furthermore, we found that early-flowering but not late-flowering gentians have a 320 bp insertion in the promoter region of GtTFL1. Thus, the negligible expression of GtTFL1 in early-flowering lines may be due to this insertion, resulting in a shortened vegetative stage.