Escherichia coli is considered as one of the critical pathogens causing contagious mastitis. The aim of the current study was 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 Jiangsu Province, China. Five hundred and thirty-five (535) milk samples presenting mastitis symptoms were collected from three dairy farms. Susceptibility testing was identified by micro-dilution method. E. coli isolates were used to detect ESBL genes (blaCTX-M, blaSHV and blaTEM) as well as COL-resistant genes mcr-(1-9). Multiplex PCR approach was used for the detection of major CTX-M groups, different phylo-groups, and virulence genes. The clonal relationship of the isolates was then evaluated with MLST, MLSA and PFGE. Multi-drug resistance (MDR) was found in more than 85% of ESBL and/or COL resistant E. coli isolates. Genotypic characterization indicated a dominance of CTX-M-1 group, and the most prevalent alleles observed in this study were blaCTX-M-28 (38.37%), blaCTX-M-14 (17.44%), blaCTX-M-66 (13.95%), blaCTX-M-55 (10.46%), and blaCTX-M-69 (6.97%), blaCTX-M-15 (4.65%), blaCTX-M-177 (4.65%), and blaCTX-M-148 (3.48%). The results confirmed that group A was the most predominant group with 54.73%, followed by group B1 and B2 (24.21%, 10.52%) respectively. The targeted virulence genes were detected in 97.89% (n=93) isolates. All isolates belong to group A, B1, B2, C, D, and E showed the presence of virulence factors. Among virulent genes ompC, was detected in 97.89% (n=93) isolates followed by fimH in 61.05% (n=58), ECs3703 57.89% (n=55), ompF 50.52% (n=48). MLST and PFGE of the 22 E. coli isolates were determined for clonal relatedness and yielded clear fingerprint patterns. Majority of the E. coli isolates carrying ESBL and mcr-1 isolates were clonally unrelated. Sequence types 58 and ST410 were the most predominant comprising 2 each (2/20=20%).