Reproductive seasonality is one of the key life-history strategies affecting fitness in nature. Some animals respond reproductively to seasonal changes such as photoperiod, while others alter or lose this seasonality. However, the molecular genetic mechanisms underlying the diversification of reproductive seasonality remain unclear. To address this question, we use the three-spined stickleback, which exhibits an incredible diversity of photoperiodic response in reproduction among various ecotypes, as a model. We recently revealed that the photoperiodic response of thyroid-stimulating hormone b2 (TSHb2) in the pituitary played an essential role in controlling the reproductive seasonality. Pituitary cells are known to be highly heterogeneous in transcriptional regulations and epigenetic modifications. In addition, our further gene-localization analysis showed that TSHb2 was only expressed in a small portion of pituitary cells. Thereby, to understand regulatory modifications during the diversification of seasonality, it is necessary to elucidate the photoperiodic response of the pituitary at a single-cell resolution. In this study, we performed simultaneous profiling of transcriptome and chromatin accessibility from single cells in the pituitary. Intriguingly, the expression of TSHb2 was concentrated in a novel cell type with distinct chromatin statuses. Overall, our findings highlighted the crucial function of TSHb2 in the diversification of reproductive seasonality.