Bacterial multidrug resistance (MDR) is a major cause of clinical failures in treating infections. According to NIH, about 70% of nosocomial infections in the U.S. are caused by strains that are resistant to at least one antibiotic. Totally, 4.5% such infections result in death, presenting a devastating threat to public health and economy. Increasing evidence suggests that bacteria can resist multiple antibiotics through intrinsic mechanisms that rely on gene products such efflux pumps that expel antibiotics and special membrane proteins that block the penetration of drug molecules. In this study, E. coli was used as a model system to explore the genetic basis of MDR. A random mutant library was constructed in E. coli EC100 using transposon mutagenesis. The library was screened to identify the mutants with enhance/reduced susceptibility to chloramphenicol. Out of the round 3000 mutants screened up to date, 15 mutants were found to be more sensitive to chloramphenicol and 8 were more tolerant. Twenty of the 23 genes responsible for these phenotypic changes were identified by inverse PCR and DNA sequencing. While 5 of the 20 genes have been reported previously as MDR genes, 15 genes were found to be related to MDR for the first time. The functions of these genes and their roles in biofilm MDR will be discussed.