Introduction Pyometra is a common and potentially life-threatening uterine infection in bitches, with severe local and systemic inflammation as symptoms. Studies exist on inflammatory markers in peripheral blood (PB), but little is known about their expression in uterine blood (UB). This study aimed to compare their concentrations simultaneously in UB and PB of bitches with pyometra, and to evaluate their diagnostic and prognostic potential. Material and Methods Twenty-two bitches with confirmed pyometra (13 closed-cervix and 9 open-cervix cases) and six healthy controls were enrolled. Blood samples were collected from the cephalic and uterine veins during ovariohysterectomy. The concentrations of IL-1α, IL-1β, IL-6 and IL-10 were measured using an ELISA validated for dogs, and C-reactive protein (CRP) was measured by an immunoturbidimetric assay. These five inflammatory markers were compared statistically between the three groups of dogs and two types of blood sample using the Mann–Whitney U and Wilcoxon tests. Results All markers were significantly elevated in affected bitches’ UB and PB. The IL-6 concentrations were significantly higher in UB than in PB, suggesting local production of this cytokine. Conversely, the IL-10 levels were higher in PB, possibly reflecting a systemic regulatory response. The concentrations of IL-1α and IL-1β differed insignificantly from each other. Those of CRP were significantly higher in open-cervix than in closed-cervix pyometra, indicating a stronger acute-phase response. Conclusion The results assign IL-6 and CRP a potential role as biomarkers for the diagnosis and prognosis of pyometra. Simultaneous analysis of uterine and systemic inflammatory responses provides insight into the disease’s pathophysiology and may support more targeted therapeutic approaches.