A total of 117 fecal samples were collected from diarrheic calves from various regions of Gujarat. Out of total fecal samples screened, 89.74 % samples were found to be positive for E. coli by PCR assay using species specific phoA gene. Region wise prevalence of E. coli were found to be highest in Surat (100%). Age wise, E. coli infection was highly prevalent in age group-I (1-10 days) i.e. 41.88%. In vitro antibiotic resistant pattern showed high resistance against pefloxacin (68.57%), oxytetracycline (65.71%) and erythromycin (64.76%) while colistin resistance was observed in 10.47% isolates. All the isolates were screened for the presence of virulence associated genes by PCR assay. Out of 105 isolates, 29.52%, 26.66% and 7.61% isolates harboured STa, F5 and LT-1gene, respectively. Whereas, 11.42%, 3.80% 2.85%, 1.90% isolates possess genes in combination of F5 and STa, F5 and LT1, STa and LT1 and F5, STa and LT-1, respectively.