Kisspeptin, the neuropeptide product of the Kiss1 gene, is critical in driving the hypothalamic-gonadal-pituitary (HPG) axis and fertility. Kisspeptin neurons are abundant in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (Arc) of the hypothalamus. These two populations mediate differential effects, with the Arc regulating negative feedback of sex steroids, and the AVPV regulating positive feedback. The latter is responsible for stimulating the preovulatory LH surge in females, under circadian control by the suprachiasmatic nucleus (SCN). Little is known about hypothalamic Kiss1 regulation during pregnancy. We aimed to characterise hypothalamic Kiss1 and Kiss1r mRNA expression in pregnant and non-pregnant mice, and investigate potential circadian regulation. Using C57BL/6J mice, brains were collected at diestrus, proestrus, and days 6, 10, 14 and 18 of pregnancy, at six time points: 8am, 12pm, 4pm, 8pm, 12am, 4am. Hypothalami were dissected into anterior (containing the AVPV) and posterior (containing the Arc) regions. mRNA levels of Kiss1, Kiss1r, Avpr1a, Rfrp and Rfrpr were analysed using real-time PCR. Analysis confirmed Kiss1 mRNA expression in the AVPV and Arc, with no indication of circadian regulation at diestrus or any pregnancy time point. During proestrus, Kiss1 expression in the AVPV increased significantly between 4pm and 8pm, coinciding with the occurrence of the LH surge. Kiss1r was detectable in both hypothalamic regions; some time-of-day variation was observed, however no clear pattern was established. Anterior hypothalamus Avpr1a expression increased at 12pm during proestrus, possibly reflecting the circadian pathway from the SCN to AVPV Kiss1 neurons. Rfrp and Rfrpr expression exhibited no circadian variability at any time point measured. Our data are the first to show Kiss1 and Kiss1r expression in the maternal brain during pregnancy. The absence of circadian regulation of Kiss1 despite high estradiol levels may indicate a disruption of the normal circadian rhythm that operates in the non-pregnant state.