| Summary: | Carbapenem-resistant Gram-negative bacilli (CR-GNB) are a major public health concern. We aimed to evaluate the prevalence of CR-GNB and the frequency of carbapenemase-encoding genes in a tertiary referral center from El Bajio, Mexico. A cross-sectional study was conducted between January and October 2022; Gram-negative bacilli (GNB) were screened for in vitro resistance to at least one carbapenem. CR-GNB were further analyzed for carbapenemase-production through phenotypical methods and by real-time PCR for the following genes: <i>bla</i><sub>KPC</sub>, <i>bla</i><sub>GES</sub>, <i>bla</i><sub>NDM</sub>, <i>bla</i><sub>VIM</sub>, <i>bla</i><sub>IMP</sub>, and <i>bla</i><sub>OXA-48</sub>. In total, 37 out of 508 GNB were carbapenem-resistant (7.3%, 95% CI 5.2–9.9). Non-fermenters had higher rates of carbapenem resistance than <i>Enterobacterales</i> (32.5% vs. 2.6%; OR 18.3, 95% CI 8.5–39, <i>p</i> < 0.0001), and <i>Enterobacter cloacae</i> showed higher carbapenem resistance than other <i>Enterobacterales</i> (27% vs. 1.4%; OR 25.9, 95% CI 6.9–95, <i>p</i> < 0.0001). Only 15 (40.5%) CR-GNB had a carbapenemase-encoding gene; <i>Enterobacterales</i> were more likely to have a carbapenemase-encoding gene than non-fermenters (63.6% vs. 30.8%, <i>p</i> = 0.08); <i>bla</i><sub>NDM-1</sub> and <i>bla</i><sub>NDM-5</sub> were the main genes found in <i>Enterobacterales</i>; and <i>bla</i><sub>IMP-75</sub> was the most common for <i>Pseudomonas aeruginosa</i>. The <i>mcr-2</i> gene was harbored in one polymyxin-resistant <i>E. cloacae</i>. In our setting, NDM was the most common carbapenemase; however, less than half of the CR-GNB showed a carbapenemase-encoding gene.
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