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Antimicrobial resistance, genotype, and virulence gene content of Escherichia coli isolated from bovine clinical mastitis in Tehran Province, Iran, were studied. Two hundred seven (207) milk samples from individual cows presenting mastitis symptoms collected from different dairy farms were used to determine the presence of specific genes of E. coli responsible for this pathology. Multiplex PCR was used to differentiate E. coli isolates into different phylogenetic groups/subgroups and to detect their virulence and involved resistance genes. All the isolated strains were tested for the susceptibility to 21 antimicrobial agents. The results showed that E. coli was detected in 42 (20.3%) samples and 69% of them belonged to the phylogenetic groups A and B1. The phylogenetic subgroup A1 (31%) and subgroup B1 (28.6%) demonstrated the highest prevalence of virulence genes (f17c‐A, and eae (n = 6), f17b‐A, and f17d‐A (n = 5), afaD‐8, afaE‐8, aucD, and bfpA (n = 4), clpG and VT (n = 2), and LT and ST genes (n = 1)). The highest antimicrobial resistance was observed for tetracycline (45.2%) followed by streptomycin (26.2%). The antimicrobial resistance genes tetB (31%), tetA (28.6%), and aadA (26.2%) were the most prevalent. Moreover, integron class 1 and 2 were found in 24 (57.1%) and 8 (19%) of the E. coli isolates. [ABSTRACT FROM AUTHOR]