Aim: To investigate the occurrence of ESBL and colistin-resistant E. coli and its genotypic characterization and identification of virulence determinants in cases of bovine mastitis in three provinces of China.Materials & methods: Five hundred and thirty-five milk samples presenting mastitis symptoms were screened for the presence of ESBL, colistin-resistant, and different virulence genes. Susceptibility testing was identified by the micro-dilution method. E. coli isolates were used to detect ESBL genes (blaCTX-M, blaSHV, and blaTEM) and colistin-resistant genes mcr-(1-9). Multiplex PCR approach was used for the detection of major blaCTX-M groups, different phylogroups, and virulence genes. The clonal relationship was then evaluated with MLST, MLSA, and PFGE. Results: Multi-drug resistance (MDR) was found in more than 85% of ESBL and colistin-resistant E. coli isolates. Genotypic characterization showed a dominance of the blaCTX-M-1 group, and the most prevalent alleles observed were blaCTX-M-28 (38.37%), blaCTX-M-14 (17.44%), blaCTX-M-66 (13.95%), and blaCTX-M-55 (10.46%). The targeted virulence genes were detected in 97.89% of isolates. Sequence types ST58 and ST410 were the most predominant (2/20=20%). The majority of the E. coli isolates carrying ESBL and mcr-1 were clonally unrelated. Conclusion: High level of association was observed between ESBL-producing and COL-resistance in E. coli of bovine mastitis.