The aetiology of primary uterine inertia (PUI), which is the most common cause of canine dystocia, is still not elucidated. Prostaglandins (PGs) play a crucial role in parturition. We hypothesized that the expression of prostaglandin endoperoxidase synthase 2 (PTGS2), PGF2α synthase (PGFS), and corresponding receptor (PTGFR) is altered in PUI. We investigated PTGS2, PGFS, and PTGFR mRNA expression, and PTGS2 and PGFS protein expression in interplacental (IP) and uteroplacental sites (UP) in bitches with PUI, obstructive dystocia (OD), and prepartum (PC). PTGS2, PGFS, and PTGFR mRNA expression did not differ significantly between PUI and OD (IP/UP). PTGFR ratio in UP was higher in PC than in OD (p = 0.014). PTGS2 immunopositivity was noted in foetal trophoblasts, luminal and superficial glandular epithelial cells, smooth muscle cells of both myometrial layers, and weakly and sporadically in deep uterine glands. PGFS was localized in luminal epithelial cells and in the epithelium of superficial uterine glands. PTGS2 and PGFS staining was similar between PUI and OD, while PGFS protein expression differed between OD and PC (p = 0.0215). For PTGS2, the longitudinal myometrial layer of IP stained significantly stronger than the circular layer, independent of groups. These results do not support a role for PTGS2, PGFS, and PTGFR in PUI. Reduced PGFS expression in IP during parturition compared with PC and the overall lack of placental PGFS expression confirm that PGFS is not the main source of prepartal PGF2alpha increase. The difference in PTGS2 expression between IP myometrial layers warrants further investigation into its physiological relevance.