In Malaysia, the aquaculture industry, particularly the production of freshwater aquaculture fish, is growing rapidly. Nevertheless, the illegal use of banned antimicrobial agents such as chloramphenicol in aquaculture has become a major concern in relation to the safety of consumers and also the development of drug-resistant strains in bacteria. Driven by those factors, the main intention of this study was to determine the prevalence and types of chloramphenicol resistance genes in E. coli isolated from aquaculture and other environmental waters. The respective chloramphenicol-resistance genes in the isolates were detected by multiplex PCR with four sense primers C-1, C-2, C-3, C-4 and one antisense primer C-R for targeting cat I, cat II, cat III and cat IV genes, respectively. Out of 27 E. coli isolated, 19 were resistant to chloramphenicol. Cat I, cat II, cat III and cat IV genes were detected in 19, 13, 10, and 6 of the E. coli isolates, respectively. The results of this study revealed that chloramphenicol-resistance E. coli is present in aquaculture and environmental waters, in the study area. This finding suggested that although banned, there could be illegal usage of chloramphenicol antibiotic in local aquaculture. The bacteria in aquaculture may have spread to other environmental water through disposal of aquaculture waste water to other environments.