Introduction Kisspeptin (KiSS), neurokinin B (NKB) and dynorphin A (Dyn A) participate in the neuroendocrine regulation of reproductive system development and functioning; however, their roles in the onset of sexual maturity and pathomechanism of delayed puberty have not yet been fully elucidated. The aim of the study was to determine changes in the kisspeptin-10 (KiSS-10), NKB and Dyn A concentration in blood plasma collected from ewe lambs during puberty in relation to the first ovulation time, and to examine the relationship between these neuropeptides. Material and Methods Twenty-four ewe lambs were divided into two groups: from single (S) pregnancies and from twin (T) pregnancies. To determine the KiSS-10, NKB and Dyn A concentration with ELISA, blood was collected from all sheep at 4, 5, 6, 7, 8, 9 and 10 months of age. Singletons and twins had their first ovulations at 8 and 10 months of age, respectively. Results Changes in the KiSS-10 and NKB concentrations were positively correlated with age in both S (r = 0.86) and T ewes (r = 0.89). A negative correlation was found between KiSS-10 and Dyn A (r = –0.55 and –0.98, for S and T, respectively) and between NKB and Dyn A (r = –0.89 and –0.94, for S and T, respectively). Conclusion The initiation of ovarian activity is associated with concurrent increases in plasma KiSS-10 and NKB concentrations with age and reduced Dyn A concentrations in both groups. Our results show that these neuropeptides may regulate the timing of reproductive activity onset in sheep.