Objective: The aim of the study is to establish a multiplex PCR for detecting some pathogenic Enterobacteriace spieces. Subject and method: This technique is based on simultaneous amplification of a plural of target gene in a single optimized reaction tube with relevant negative and/or positive controls. Detection limits was determined by piking assays in a range from 104 + 10 CFU/ml. Result: the data demonstrated that the selected target gene fragmnents are suitable for calling up Escherichia coli, Klebsiella pneumoniae, Salmonella sp, Proteus sp, Enterobacteriaceae sp from clinical samples. Specificity and sensitivity of endpoint PCR were 94-99 percent. Conclusion: A multiplex PCR for detecting mentioned pathogens was established with a detection threshold of 10 CFU/ml and 94-99 percent specificity.