Kisspeptin, the neuropeptide product of the Kiss1 gene, has a critical role in driving the hypothalamic-gonadal-pituitary (HPG) axis and reproductive function. Kisspeptin neurons are most abundant in the hypothalamus, particularly in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (Arc). 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, and is consequently under circadian control. Little is known about the role of kisspeptin or hypothalamic Kiss1 regulation during pregnancy. We aimed to characterise hypothalamic Kiss1 and Kiss1r mRNA expression during pregnancy in the mouse, and determine whether it is under circadian control. Using C57BL/6J mice, maternal brains were collected at days 14 and 18 of pregnancy, at six circadian time points: 8am, 12pm, 4pm, 8pm, 12am, 4am. Hypothalami were dissected into anterior (containing the AVPV) and posterior (containing the Arc) regions. mRNA levels for target genes Kiss1 and Kiss1r, as well as reference genes (SDHA, HPRT) were analysed using real-time PCR. Analysis confirmed Kiss1 mRNA expression in the AVPV and Arc; quantitative assessment showed no overall change in expression from day 14 to 18 of pregnancy and no clear indication of circadian regulation (AVPV P=0.39; Arc P=0.29). Kiss1 levels appeared greater in the AVPV compared to the ARC, which is consistent with the high levels of circulating estradiol and progesterone in late pregnancy. Similarly, Kiss1r was detectable in both the anterior and posterior hypothalami, but again no circadian pattern of expression was observed. Overall, our data are the first to show Kiss1 and Kiss1r expression in the maternal brain late in pregnancy. The absence of circadian regulation of Kiss1 or Kiss1r may indicate a disruption of the normal circadian rhythm that operates in the non-pregnant state.