OBJECTIVE: The epidemiology of Extended-spectrum beta-lactamase (ESBL) producing E coli and K pneumoniae is complex and varies among hospitals and countries. This study aimed at describing the molecular detection and epidemiology of ESBL subtypes prevalent in clinical isolates of K pneumonia and E coli in Trinidad and Tobago. METHODS: Over 36-months, isolates of E coli and K pneumoniae from clinical specimens of patients processed at a regional tertiary hospital in the country,were identified using standard microbiological methods. MicroScan System (Siemens, USA) was used to determine MIC values while E-test (AB Biodisk, Sweden) assays phenotypically confirmed ESBL production. K pneumoniae (n = 65) and E coli (n = 25) isolates confirmed as ESBL producers were further subjected to multiplex PCR and PFGE tests to determine the ESBL subtypes and clonal relatedness. RESULTS: Female patients (67.8%) and urine samples (65%) yielded most ESBL isolates, with over 90% recovered from the hospital's medicine and surgery facilities. All ESBL isolates including all K pneumoniae producing ESBLs were 100% susceptible to carbapenems and amikacin antimicrobials. Polymerase Chain Reaction detected 100% blaTEM genes, 4.1% blasHv and 37.5% blaCTX_M genes among E coli isolates. Similarly, 84.3% blaTEM, 34.5% blaSHV and 58.8% blaCTX-M genes were detected in K pneumoniae. Pulsed-field gel electrophoresis (PFGE) results showed diverse and unrelated clones. CONCLUSIONS: In this the first report of molecular characterization and epidemiology ofESBL subtypes in E coli and K pneumoniae isolates in Trinidad and Tobago, the CTX-M, mainly phylogenetically group 1 type, was most predominant. Most ESBL isolates were still susceptible to carbapenems and aminoglycosides and their spread appears to be polyclonal and clonally unrelated.