Escherichia spp. is reported as the major important environmental agent causing mastitis in lactating cattle herds. Because of its wide distribution and rapped dissemination all over the herd animals, accompanied by high economic loses. This study was conducted for detection and determination of the resistance genes in E. coli strains isolated from milk of cattle suffering mastitis. The incidence rate of E. coli infection, (4.6%), represented by 8 isolates/175 milk samples. E. coli isolated strains were identified by applying the traditional methods of isolation and identification, following confirmation by PCR, that reporting the amplification of 16S rRNA (phoA gene) at 720 bp. On the identified isolates, the antibiotic sensitivity test was carried out against 6 antibiotics that belong to 6 antibiotic groups; ciprofloxacin (quinolones), erythromycin (macrolides), streptomycin (aminoglycosides), norfloxacin (fluoroquinolones), clindamycin (lincosamides) and sulphamethoxazol+ trimethoprim (sulphonamides). The results showed that the isolates had complete resistance (100%) to erythromycin (macrolides), streptomycin (aminoglycosides), clindamycin (lincosamides) and sulphamethoxazol + trimethoprim (sulphonamides). The PCR detected presence of 4 resistance genes sul1, mphA, aadA1 and ereA, in all tested isolates, while dfrA resistance gene, was detected in 5 isolates only (62.5%). This study concluded that E. coli isolated from milk of cattle suffering mastitis showed multidrug resistance activity and carried several resistance genes, which playing an essential role in the resistance activity effect toward the antibiotic drugs of choice for treatment of diseased animals.