Antibiotic microbial resistance is a major health problem worldwide in both humans and animals, specifically, E. coli producing extended-spectrum β-lactamases (ESBL). In the present study, E. coli was isolated from human; broiler; and calves samples, and then the isolates were biotyped. To determine E. coli antibiotic sensitivity pattern, isolates were subjected to antibiogram. Consequently, seven of the multi-drug resistant isolate were randomly selected and subjected to polymerase chain reaction to detect ESBL. The isolates showed high degree of resistance to beta-lactame antibiotics. Among the seven isolates, four isolates were TEM enzyme producer; 3 isolates were SHV producers; and another 3isolates were CTX-M producers. Point mutation was detected at different positions in TEM, SHV, and CTX genes as detected by DNA sequencing which releaved point mutation in TEM gens at position number (89,90,114,115,116,135,485,1077 and1078) while SHV gens at position number (114,237.643and 635) and CTX-M gens at position number (364,382,383,707,708,736,737,286 and287), in isolates of human, claves and broilers. It could be conclude that the major reason behind E. coli antibiotic resistance is the production of ESBL and that genes undergo mutation at different sites in different species.