Objective: To investigate the relatedness in 60 drug-resistant Escherichia coli(E.coli) strains. Methods: Drug-resistance related genes were analyzed by PCR,including 4 kinds of housekeeping genes and 45 kinds of horizontal transfered genes related to 3 kinds of antimicrobial agents:beta-lactams,aminoglycosides,quinolones,as well as 7 kinds of genetic markers of integrons,transposons,insertion sequences and conjugal plasmids. They were used for the sample clustering analysis. Results: Polymorphism were distinct in 60 E.coli strains. Most strains showed evolution. Only 3 strain groups which consisted of strain 6 and 53,strain 10 and 14,strain 7,8,26 and 40,were the same clones carried the same genes. Conclusion: Sample clustering analysis of drug-resistance related genes could distinguish the strains with the same antimicrobial resistance characteristics,which was significant in epidemiologic research and provide convenience for tracing transmissible routes of drug-resistant strains.