In many species, sexual activity varies on a seasonal basis. Kisspeptin (Kp), a hypothalamic neuropeptide acting as a strong activator of GnRH neurones, plays a critical role in this adaptive process. Recent studies reported that two other neuropeptides, namely neurokinin B (NKB) and dynorphin (DYN), are co-expressed with Kp (and therefore termed KNDy neurones) in the arcuate nucleus and these peptides are also thought to influence GnRH secretion. The aim of this study was to establish whether hypothalamic NKB and DYN expression is photoperiod-dependent in a seasonal rodent, the Syrian hamster, which exhibits robust seasonal rhythms in reproductive activity. The majority of Kp neurones in the arcuate nucleus co-express NKB and DYN and expression of all three peptides is decreased in short, compared to long, photoperiod leading to a 60% decrease in the number of KNDy neurones in photo-inhibitory conditions. In seasonal rodents, RFRP neurones of the dorsomedial hypothalamus are also critical for seasonal reproduction. Interestingly, NKB and DYN are also expressed in the dorsomedial hypothalamus but do not co-localise with RFRP-immunoreactive neurones, and expression of both NKB and DYN is higher in short photoperiod, which is opposite to the short day inhibition of RFRP expression. In conclusion, this study shows that NKB and DYN display different photoperiodic variations in the Syrian hamster hypothalamus. In the arcuate nucleus, NKB and DYN, together with Kp are down-regulated in short photoperiod, whereas in the dorsomedial hypothalamus NKB and DYN are up-regulated in short photoperiod. This article is protected by copyright. All rights reserved.