Bacteria have been traditionally classified on the basis of their morphology, biochemical reaction, serology and etc. However, in some case these methods could not authenticate the closely related bacteria strains. In this study, we cloned two repetitive DNA sequences with 0.9 and 0.6 kb in length from Escherichia coli K12 strain JM109 and designated as ECR-1 and ECR-6 respectively. Using ECR-1 and ECR-6 sequences or their combination as the probes for DNA polymorphism analysis, we were be able to develop a molecular method of biotyping for the identification of very closely related strains of Escherichia coli. Both of ECR-1 and ECR-6 probes could be applied for the taxonomy, epidemiological and microecological studies, and clinical diagnosis for pathogenic Escherichia coli strains.