Chrysanthemum (Dendranthema grandiflorum Kitamura), a qualitative short-day plant, does not produce flowers when the night length is shorter than a cultivar-dependent critical value. Flowering is effectively inhibited when the required uninterrupted night period is broken by short periods of exposure to night interruption lights (NILs). The effects of the quality and positioning of the NIL on the morphogenesis, flowering, and photoperiodic gene expressions of chrysanthemum were investigated. Blue (NI-B), red (NI-R), far-red (NI-Fr), and white (NI-W) light-emitting diodes (LEDs) at an intensity of 10 μmol m −2 s −1 photosynthetic photon flux density (PPFD) were used to interrupt the dark period or plants grown in a closed walk-in growth chamber at night for a total of 4 h. The NILs were positioned to illuminate either the shoot tip, youngest mature leaf (vigorously growing third leaf from the shoot tip), or old leaf (third leaf from the stem base). The NI-R on the shoot tip and all NI-Fr treatments increased the plant extension growth, to a similar height observed in plants in long-day (LD) treatments. Extension growth was generally suppressed for plants in the NIB , and plant height was the smallest when NIB was shed onto the old leaf. Plants in all NIL treatments except NI-Fr showed a negative correlation between the plant height and the age of the leaf (or tissue) exposed to the NIL. Flowering was observed for plants in all treatments except for NI-R shed onto the shoot tip. The transcriptional factor PHYA was slightly expressed in plants which showed a flowering response. However, the CRY1, PHYB, and TFL was not expressed to levels reported previously, and the reason for low expression of these genes in these plants may be due to a combined effect of these genes with other flowering promoting and/or inhibiting genes. These results suggest that both the NIL quality and the age of the leaf (or tissue) exposed to the NIL affect the morphogenesis, flowering, and expression of transcriptional factors. Moreover, the previously known effects of the NIL quality on the morphogenesis as well as the vegetative to reproductive phase transition of plants were the most pronounced when the NIL irradiated the shoot tip, and the least pronounced when the NIL was shed onto the old leaf. Therefore, it seems that not only the appropriate quality, but also the appropriate positioning of the NIL, is important for optimizing flowering and plant extension growth.