The present study was conducted on 44 commercial broiler chicken farms (20-30 days old) located in four different centers at Kaliobia Governorate ( Benha, Kafer-Shoker, Toukh and Shebin El-kanater), 11 from each were inspected for E. coli infection. Samples were taken from diseased and freshly dead chickens (liver, heart blood, lung, intestine, kidney and spleen from each chicken) for bacteriological examination. The results revealed that 502 out of 1320 samples (38%) were positive for E. coli isolation, where 124 isolates (24.7%) from 408 samples of 68 diseased chickens and 378 isolates (75.7%) from 912 samples of 152 freshly dead chickens. Three seogroups of E. coli were obtained by serological identification (055, 0125, and recently 0146), represented as 6 strains were serotyped 0146 (40%), 3 stains 0125 (20%), 2 strains 055 (13.3%) and 4 isolates were untyped by the available antisera. Moreover, the serogroups 055 and 0146 were positive for K99 while 0125 was negative. Gentamycin, Cefotaxime, Ampicillin / Clavulinic acid and Enrofloxacin were the most proper antibiotics with the highest in vitro efficiency against isolated E.coli. Finally, Multiplex PCR showed that eaeA, ompA, kpsMTII, tsh, iutA and iss virulence genes were detected in all serogroups. While papC virulence gene was detected in serotypes 055 only. Moreover, stx2 virulence gene was detected in both serotypes 055 and 0125, meanwhile, it was not amplified in 0146 serotype