The aim of the present study was to determine the concentrations of glutathione (GSH), vitamin C, copper (Cu) and zinc (Zn) in the uterine tissues in diagnosis of canine pyometra. Fourteen samples of uterine tissues from female dogs with pyometra and twelve samples of healthy uteruses (control) were used. The concentrations of GSH and vitamin C were determined in the uterine tissue homogenates using spectrophotometric methods. The concentrations of Cu and Zn were measured using atomic absorption spectrometer. The results obtained showed the significantly lower (p<0.05) concentration of GSH and the trend towards lower concentration of vitamin C in the pyometra samples compared to the control. The concentrations of Cu and Zn were similar in the uterine tissues from female dogs with pyometra and those from healthy female dogs. The lower GSH and vitamin C concentrations in the uterine tissues of female dogs with pyometra indicate that the non-enzymatic antioxidant mechanisms are impaired in the uterus of dogs with pyometra. These findings suggest that the imbalance of oxidative-antioxidative can play an important role in pathogenesis of canine pyometra.