Background: Canine pyometra, is a pathological condition that results in the accumulation of purulent exudate in the uterus due to hormonal imbalance in female dogs. This is the most prevalent hormonally mediated reproductive disorder and life threatening disease of intact female dogs. The objective of the present study was to assess the physiological, haematological and biochemical changes and isolate the pathogenic microorganism present in the uterus of pyometra bitches. Methods: This study was carried out in ten bitches with a history of lethargy, depression, in appetence, polydipsia, occasional vomiting and vaginal discharge presented for treatment at the Veterinary Poly Clinic, Coimbatore, Tamil Nadu. The disease was diagnosed by history, clinical signs, physical examination, imaging techniques haematology and biochemical study. The involvement of the pathogenic organism was confirmed by microbial examination of the pus-filled uterus after the ovariohysterectomy. Result: The result showed that physiological parameters rectal temperature, heart rate and respiration rate were increased in pyometra affected dog than healthy one. The haematological parameters of red blood cell count, haemoglobin and packed cell volume were decreased in pyometra affected dogs than healthy dogs. Leukocytosis, neutrophilia, lymphocytosis, monocytosis and eosinophilia condition also observed in pyometra affected dogs. Biochemically, increased Blood urea nitrogen (BUN) creatinine and hyperprotenimia were observed in pyometra dogs. Increased activity of hepatic enzyme Alanine transaminase (ALT), Aspartate aminotransferase (AST) and Alkaline phosphatase (ALP) were also observed in pyometra dogs. The major pathogenic microorganisms involved in pyometra are Escherichia coli (40.25%), Staphylococcus (20.46%), Streptococcus (20.32%), Pseudomonas (6.41%), Proteus (6.32%) and Pasteurella (6.24%). Thus the dogs with pyometra show significant changes in physiological, haematological, biochemical parameters and growth of pathogenic microorganism in uterine fluid were the important indications of pyometra affected dogs.