Timing of breeding is under selection in wild populations due to climate change, and understanding the underlying physiological processes mediating timing provides insight in the potential rate of adaptation. Current knowledge on this variation in physiology is, however, mostly limited to males. We assessed whether individual differences in timing of breeding in females are reflected in differences in candidate gene expression and if so, whether these differences occur in the upstream (hypothalamus), or downstream (ovary and liver) parts of the neuroendocrine system. We used 72 female great tits from two generations of lines artificially selected for early and late egg-laying, which were housed in climate controlled aviaries and went through two breeding cycles within one year. In the first breeding season we obtained individual egg-laying dates, while in the second breeding season, using the same individuals, we sampled several tissues at three time points based on timing of the first breeding attempt. For each tissue, mRNA expression levels were measured using qPCR for a set of candidate genes associated with timing of reproduction and subsequently analysed for differences between generations, time points and individual timing of breeding. We found differences in gene expression between generations in all tissues with most pronounced differences in the hypothalamus. Differences between time points, and early and late laying females, were found exclusively in ovary and liver. Altogether, we show that fine-tuning of seasonal timing of breeding, and thereby the opportunity for adaptation in the neuroendocrine system, is regulated mostly downstream in the neuro-endocrine system.