High temperature-induced delay of flowering is a major problem during the production of short-day (SD) plants such as Chrysanthemum.We have investigated the heat-sensitive hours for flowering in C. seticuspe. Heat treatment at 30°C was applied for 16, 8, or 4 h d-1, at different times of the day, under SD conditions (8 h photoperiod from 08.00 - 16.00 h), using 20°C as the optimum temperature. Heating between midnight and dawn (00.00 - 08.00 h) delayed floral initiation for up to 2 d, but delayed flowering for > 25 d, which represented the heat-sensitive hours for capitulum development. Heating during this period suppressed expression of the FLOWERING LOCUS T-like3 (CsFTL3) gene, a promoter of flowering induced in leaves by SDs. However, expression of CsFTL3 was higher compared to non-floralinductive photoperiods with a night-break. The anti-florigenic FT/TFL1 family protein gene (CsAFT) is an inhibitor of flowering induced in leaves by long-days. Expression of CsAFT remained low during heating under SDs, even when the plants flowered late. Our results show that high temperatures had little effect on floral initiation, which required suppression of CsAFT and a slight induction of CsFTL3 gene expression under SDs. In contrast, a high temperature between 00.00 - 08.00 h appeared to delay flowering by decreasing the level of expression of CsFTL3 required for capitulum development.