Abstract: Three hundred random samples were collected from different localities in Assiut city including raw milk (150) and some dairy products as Kareish cheese, Domiati cheese, Ras cheese, small scale producers ice cream and street vendors ice cream (30 samples for each). These samples were examined for the incidence of C. diphtheria using two selective media: Mueller-Hinton medium with Rabbit serum and Medium D2. The incidence of C. diphtheriae was (0.9%) and (3.5%) on Mueller-Hinton medium with Rabbit serum and Medium D2, respectively. Comparison between two selective media used for isolation of C. diphtheria proved the superiority of Medium D2 in the isolation of C. diphtheria from milk and some milk products. Polymerase Chain Reaction (PCR) was done to 8 C. diphtheria strains previously identified by biochemical tests and negative results were obtained. These strains may be non toxogenic or may be genetically varied. So we suggest making genetic sequencing for the isolated C. diphtheria strains and creating specific primers for locally isolated strains. More efforts are needed to enhance and promote farms and sale points of milk.