Abstract: An investigation on occurrence of Enterohaemorrhagic E. coli in beef was carried out to determine the prevalence rate in Thrissur. A total of 175 fresh beef samples were collected four retail outlets and two slaughter houses located at Thrissur. Hundred gram of the material was taken from each sample. The isolation and identification was carried out by FDA method with necessary modification. Five samples were positive for Enterohaemorrhagic E. coli and virulence gene stx 1 was obtained from two isolates using polymerase chain reaction. None of the isolates was positive for stx 2 and eae A genes.