Introduction. Lack of laboratory capacity hampers consistent national antimicrobial resistance (AMR) surveillance. Chromogenic media may provide a practical screening tool for detection of individuals colonized by extended-spectrum beta-lactamase (ESBL)-producing organisms. Hypothesis. CHROMagar ESBL media represent an adequate screening method for the detection of extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE), isolated from rectal swabs. Aim. To evaluate the performance of CHROMagar ESBL media to accurately identify ESCrE isolates from rectal swab samples attained from hospitalized and community participants. Methodology. All participants provided informed consent prior to enrolment. Rectal swabs from 2469 hospital and community participants were inoculated onto CHROMagar ESBL. The performance of CHROMagar ESBL to differentiate Escherichia coli and Klebsiella spp., Enterobacter spp. and Citrobacter spp. (KEC spp.) as well as select for extended-spectrum cephalosporin resistance were compared to matrix-assisted laser desorption/ionization-time-of-flight MS (MALDI-TOF-MS) and VITEK-2 automated susceptibility testing. Results. CHROMagar ESBL had a positive and negative agreement of 91.2% (95% CI, 88.4–93.3) and 86.8% (95% CI, 82.0–90.7) for E. coli and 88.1% (95% CI 83.2–92.1) and 87.6% (95% CI 84.7–90.2) for KEC spp. differentiation, respectively, when compared to species ID by MALDI-TOF-MS. When evaluated for phenotypic susceptibilities (VITEK-2), 88.1% (714/810) of the isolates recovered on the selective agar exhibited resistance to third-generation cephalosporins. Conclusion. The performance characteristics of CHROMagar ESBL media suggest that they may be a viable screening tool for the identification of ESCrE from hospitalized and community participants and could be used to inform infection prevention and control practices in Botswana and potentially other low-and middle-income countries (LMICs). Further studies are required to analyse the costs and the impact on time-to-result of the media in comparison with available laboratory methods for ESCrE surveillance in the country

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