Milk quality continues to be a topic of intense debate in the dairy industry, medical and public health communities. Production of maximum quantities of high-quality milk is an important goal of every dairy operation. High-quality milk must contain a low number of somatic cells and low bacteria count, and must be free of human pathogens and antibiotic residues. The objective of this study was to determine the prevalence of E. coli in unpasteurized milk recovered from Middledrift and Fort Hare dairy. In this study 400 milk samples were collected from two commercial farms (Middledrift and Fort Hare) in the Eastern Cape, South Africa, 200 raw milk samples from each farm. Samples were cultured on violet red bile mug-agar (VRB-MUG Agar) and incubated at 37ºC for 24 hours and preliminary identified by Gram stain and catalase test. Isolates that were Gram negative and catalase positive were screened for a marker of E. coli uidA gene using PCR assays. Middledrift dairy farm had 50 (25%) E. coli isolated from raw milk and Fort Hare farm showed 37 (18.5%) E. coli present in the milk samples. The presence of E. coli found in the milk samples points to the fact that fecal contamination was unavoidable and traditional practices are likely to contribute to the contamination of the milk and proliferation of the microorganisms.