Background <p>The Xpert Carba-R assay allows direct identification of major carbapenemase genes from stools, offering faster results than culture-based methods. This study aimed to evaluate the diagnostic performance of the Xpert Carba-R assay for detecting CPE colonization and assess gene-level concordance between direct stool and cultured samples.</p> Methods <p>Between March and December 2023, two rectal swabs were collected from high-risk patients, those admitted to the intensive care unit or with prior hospitalization—at a tertiary care hospital. One swab was analyzed using the direct stool Xpert Carba-R assay, and the other underwent conventional culture techniques. Diagnostic performance was evaluated against culture-based detection, and concordance of carbapenemase gene identification between direct stool and culture isolate results was assessed.</p> Results <p>Among 4,120 screened patients, 107 (2.5%) were colonized with CPE. The direct stool Xpert assay showed a sensitivity of 97.2% and specificity of 99.1% for CPE detection. Concordance analysis showed an overall agreement of 64.1% (Cohen’s kappa coefficient = 0.456). Discordance occurred in 52 cases (35.9%), primarily due to multiple gene detections in direct stool samples that were not confirmed in cultural isolates. The positive predictive value varied significantly by gene: <i>bla</i>_KPC showed 90.8%, <i>bla</i>_NDM 52.6%, <i>bla</i>_OXA-48 50.0%, while <i>bla</i>_IMP-1 showed 0%, with none of the nine <i>bla</i>_IMP-1 detections from stool samples confirmed in corresponding cultured isolates.</p> Conclusion <p>The Xpert Carba-R assay is a reliable tool for direct CPE detection from stool samples, though discordance with culture-based testing, particularly for <i>bla</i>_IMP-1—should be considered in clinical interpretation.</p>