A total of 400 Recto-anal mucosal swabs samples (200 from each region) were collected and processed initially by conventional PCR. Among them, only two (n=2) RAMS samples were detected and positive for E. coli O157 gene, included one in RAMS samples of sheep from slaughterhouse of Rawalpindi, while another in RAMS samples of buffalos from slaughterhouse of Islamabad. The rest of the samples from goats and cows showed negative results for E. coli O157 in both regions. E. coli O157 isolates were obtained from each positive sample onto two different Sorbitol MacConkey Agar media plates and analyzed again through conventional PCR, resulted only single 1/6 isolated colony con rmed from both samples which possessed rfbE genes. The overall prevalence of rfbE (O157) genes came up to (0.5%). Furthermore, for the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA), these both positive rfbE isolated colonies were then brie y subjected towards multiplex PCR. Results revealed that none of the STEC virulent genes were detected in single (01) positive rfbE O157 isolated colony of sheep sample. While fortunately 4 STEC virulent genes (sxt1, stx2, eae and ehlyA) were detected in single (01) positive rfbE O157 isolated colony of buffalo sample. It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, buffalo sample possessed 4 STEC virulent genes, hence considered an important source of STEC infection to humans either through direct or indirect transmission which should devise proper control systems.