Pyometra is one of the most common uterine pathologies of intact bitch at middle to advanced age. In the early stages, the disease shows subtle changes, making diagnosis a challenge. In contrast, at later stages, it manifests as potentially life-threatening systemic inflammatory response syndrome. Ultrasonographic examination of the uterus aids in the diagnosis, although it has limitation in ascertaining the clinical severity of pyometra. Moreover, differentiation of cystic endometrial hyperplasia from pyometra could not be disced with greater accuracy. Therefore, false negative diagnosis of pyometra patients leads to development of systemic inflammatory response, which delays administration of therapies and results in deaths during early course of treatment. Further, indiscriminate use of broad-spectrum antimicrobials at higher dose in false positive cases considerably contributes to the rising pool of drug resistant pathogens, thereby increasing the risk of case fatality due to sepsis in a long-term. Monitoring the circulating pro-inflammatory cytokines, acute phase proteins, endotoxin, growth factors and inflammatory mediators is the current trend in pyometra research, especially for developing diagnostic and prognostic biomarkers. The present review deals with the prospects of developing diagnostic and prognostic biomarkers in the canine pyometra.