Uterine tissue was collected from bitches after ovariohysterectomy at different times after ovulation. Samples were assigned to four groups: metestrous non-pregnant, day 10–12, n = 4; pre-implantation, day 10–12, n = 9; post-implantation, day 18–25, n = 13; mid-gestation, day 30–40, n = 7. RT-qPCR detection was performed for kiss1 and the G protein-coupled receptor 54 (GPR54, specific receptor for kisspeptin). In addition, immunohistochemistry was performed for detection of kisspeptin-10 (KP-10), GPR54, as well as pan-cytokeratin and vimentin. The latter two were included to differentiate the different placental cell types. The percentage of positive stained cells was evaluated, and an immunoreactivity score (IRS) was obtained by multiplying the labelling intensity score (0–3) with the percentage of immunolabelled cells (range: 0–300). In non-pregnant and pre-implantation tissues, gene expression was highly variable for kiss1 and GPR54. Expression of GPR54 was higher before embryo adhesion than during post-implantation and mid-gestation (p < .05), whereas there was no difference found between groups for kiss1. Except during the pre-implantation period, KP-10 expression was higher in the non-pregnant uterus compared to all gestational periods investigated, indicating a pregnancy-related downregulation. In the pre-implantation period, KP-10 was present in larger vessels only, whereas the presence of GPR54 in vessels was found in all samples, with most labelling in the post-implantation period. KP-10 was present in superficial uterine glands, GPR54 in superficial and deep uterine glands of the post-implantation uterus. In myocytes, the highest staining for KP-10 was seen in the non-pregnant uterus, whereas the highest staining for GPR54 was seen in post-implantation and mid-gestation. Syncytiotrophoblast cells stained for both KP-10 and GPR54 in post-implantation and mid-gestation, with maximum intensity for GPR54 in the latter. We conclude that KP-10 and GPR54 are expressed in the canine uterus and trophoblast cells. However, during pregnancy, expression of both proteins seems to be differentially regulated.