Colibacillosis is a fetal and serious problematic disease particularly in young calves during the first days of their life accompanied with high mortality losses. The aim of this study is to moleculary characterize Escherichia coli (E. coli) strains isolated from individual cases of neonatal calf diarrhea. Characteristic clinical symptoms as yellowish diarrhea with low colostrum feeding history was observed during samples collection. Rectal swabs were obtained from 50 healthy and 100 clinically infected diarrheic calves which identified by traditional bacteriological and serological procedures. Then, E. coli isolates were subjected for genetic characterization of virulence genes (stx1, stx2, fimH, iutA, eaeA and tsh) using both uniplex and multiplex PCR. The most dominant virulence gene was fimH in all the examined isolates followed by iutA in 50% however, the eaeA and tsh genes were in 37.5% while no detection for both stx1 and stx2 genes. In conclusion, these results provide updated information regarding the molecular detection of E. coli strains from neonatal calves in Egypt and thus, would be important to formulate preventing tools and effective therapy against E. coli either in Egypt and or worldwide.