Molecular techniques were used for studying the epidemiology of Escherichia coli from five major towns in Ondo state, namely; Akure and Ondo, Owo, Okiutipupa and Ikare-Akoko. In total, 807 urine and faecal samples of apparently healthy primary school pupils were gathered between December 2012 and March 2013. Withal, 206 E. coli strains were isolated and characterized by biochemical tests. Polymerase chain reaction (PCR) assay was performed to determine the presence of the genes-encoding virulence factors. A total of 22 (12 male, 10 female) isolates out of 28 subjected to PCR amplification were found to possess only stx1 virulence gene associated with Enterohaemorrhagic E. coli strains. PCR assay was demonstrated to be a useful technique for the epidemiological work of E. coli where this organism is a major cause of infection.