Abstract: Background and Objective: Throughout the world, antibiotic resistance has been recognized as an emerging major issue of concern. In this study, genes responsible for the production of extended spectrum beta-lactamase (ESBL) in various E. coli isolates were characterized. Materials and Methods: Isolates of E. coli were obtained from clinical and environmental samples. These samples were collected from Cape Coast, Ghana. The isolates were tested for ESBL production. Subsequently, PCR assays of DNA extractions from the isolates were used to determine prevalence of blaTEM and blaSHV genes. Results: Findings revealed that more ESBL producers were phenotypically identified in E. coli isolates recovered from clinical than those recovered from environmental samples. Very low percentages of environmental and clinical isolates that were phenotypically confirmed as non-ESBL producers were found to carry only blaTEM. There was no detection of either gene in majority of the environmental and clinical isolates that were phenotypically confirmed as non-ESBL producers. Conclusion: The environment could be a repository of blaTEM and blaSHV genes thus it is imperative to intensify public health education on the risks associated with the misuse of antibiotics.