Colibacillosis is a neglected bacterial disease affecting neonate lambs and leading to severe diarrhea and high mortality. This study aimed to diagnose Escherichia coli in diarrheic lambs and determine the level of antibiotic resistance, and identify antibiotic resistance genes (ARGs). Thirty faecal and blood samples were gathered from diseased lambs in the southwest of Basrah. The faecal samples were initially subjected to the bacteriological test, disc diffusion test, and molecular method, while blood samples were haematologically analysed. The molecular test utilized 16sRNA with the specific primers targeted the genes resistance. All samples in the selective media were initially found to be positive to E. coli. Based on the antibiotic-sensitivity results, colonies were sensitive to all antibiotics tested, with bacterial inhibition zones have varied degree. Ciprofloxacin, norfloxacin, and chloramphenicol showed 40% of susceptibility rate, while gentamycin and amoxicillin was 60%, 50%, respectively. Polymerase Chain Reaction (PCR) of 16sRNA unfold the amplification of DNA products at 1500 bp for ten isolates. The PCR amplification also identified class 1 integron, with the bands were detected at 160 bp. The haematological analyses indicated that all infected lambs were having high level of leukocytes, whereases the erythrocyte indices remained unchanging. Also, the level of comprehensive metabolic panel (CMP) did not alter by infection. The liver, enzymes, Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) were elevated during the infection. In conclusion, E. coli is a major pathogen in neonatal lambs, showing variable antibiotics resistance is linked to the resistance gene.