We aimed to investigate the occurrence, phylogeny, and virulence of E. coli in the uterine contents and urine of female dogs with pyometra, through the presence of virulence genes and their genetic similarity. Uterine secretions and urine samples from 52 female dogs with pyometra were collected and cultured. Strains identified as E. coli from 25 uterine and 7 urine samples were tested for virulence genes by PCR. Genetic similarity between the isolates was studied using RAPD-PCR. E. coli was observed in 48.07% uterine samples with pyometra and 20.0% urine samples. The strains showed high percentages for the presence of virulence genes: 96.9% had the gene sfa, 59.4% afa, 46.9% pap, 53.1% hly, and 68.75% cnf. Even with the high prevalence of virulence genes, the samples were not submitted to DNA sequencing to confirm the results. Analysis showed high genetic diversity in E. coli, however, strains isolated from the same animal indicate that cystitis and pyometra could be related. Our study indicated the association between E. coli in dogs with pyometra and cases of urinary tract infection and the pathogenic potential of strains increasing with animal age.