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The present study investigates prevalence, antibiotic susceptibility pattern and molecular characterization of multi drug resistant uropathogenic Escherichia coli isolated from patients. Antibiotic susceptibility testing (AST) revealed a significantly high resistant level in all fifty uropathogenic E. coli (UPEC) isolates against tested antibiotics (tetracycline, doxycycline, azithromycin, erythromycin, ciprofloxacin, levofloxacin, ampicillin, amoxicillin, amikacin and gentamycin) and was found as multidrug resistant. The amplified products of housekeeping genes (adk, gyrB, icd, mdh, purA, recA and fumC) and 16S rRNAgene of E. coli isolates were subjected to Restriction fragment length polymorphism (RFLP) analysis and correlated with antibiotic susceptibility pattern. Using this molecular marker technique, it was evident that genetic variability occurred among E. coli isolates from different infectious samples. In AMOVA analysis, the calculation of Nei’s genetic diversity (H) and Shannon’s information index(I) were observed maximum in group-I of housekeeping genes (H-0.2032, I-0.3265) thereby confirming remarkable polymorphisms in group-I isolates. The dendrogram obtained from Neighbor-joining and STRUCTURE analysis revealed splitting of isolates into three clusters with clear distinction of sensitive/ low resistance strains from moderate resistance and high resistance strains. This study reports the clear correlation between genetic and phenotypic aspects of E. coli isolates which is quite evident from statistical analysis like AMOVA, structural analysis and Principal Coordinate analysis. The data obtained from this study could be useful in designing strategies for better management of UTI disease.