Abstract:[Objective] We characterized the uterine microbiota in healthy felines and felines with pyometra, aiming to reveal the effect of pyometra on the uterine microbiota of felines and explore the potential pathogens causing pyometra. [Methods] High-throughput sequencing of the full-length 16S rRNA gene was employed to determine and compare the uterine microbiota in healthy felines and felines with pyometra. The key strains were isolated and identified by the culture method. [Results] The dominant bacterial genera in the uterus of healthy felines were Acinetobacter, Pseudomonas, Sphingomonas, and Weissella. The dominant bacterial genus and species in the uterus of felines with pyometra were Escherichia-Shigella and Escherichia coli, respectively. Functional prediction showed that pathways such as protein export, amino acid-related enzymes, protein processing in endoplasmic reticulum, and aminoacyl tRNA biosynthesis in the pyometra group were significantly reduced. The results of isolation and identification showed that the prevalent bacterial species in the uterus of felines with pyometra was E. coli. The isolates all belonged to the phylogroup B2 and were mostly tested positive for hylA, fimH, iroN, cnf1, papC, kpsMTII, and iutA. [Conclusion] We compared the uterine microbiota in healthy felines and felines with pyometra. The dominant bacteria in the uterus of healthy felines were mostly non-pathogenic, while those in the uterus of felines with pyometra changed significantly, with E. coli being dominant and carrying multiple virulence genes. The findings provide a theoretical basis for treating pyometra in felines.