OXA-181 is the second most common global OXA-48-like carbapenemase and is endemic in the Indian subcontinent. Molecular studies have shown that Enterobacterales with OXA-181 are often introduced into regions of nonendemicity. Distinguishing OXA-181 from other OXA-48-like enzymes often requires sequencing, which is rather expensive and time-consuming. A specific PCR (i.e., OXA181PCR) for the detection of blaOXA-181 was validated using a global collection (n = 315) of bacteria with well-characterized carbapenemases and showed 100% sensitivity and specificity (95% confidence interval [CI], 94.1 to 100 and 98.6 to 100, respectively) for detecting bacteria with OXA-181. The OXA181PCR subsequently gave positive results on 58/160 (36%) Enterobacterales with OXA-48-like carbapenemases from the 2015 INFORM surveillance program. The blaOXA-181-positive Enterobacterales were present in 9 countries spanning 5 continents, illustrating the global distribution of OXA-181. This methodology can easily be incorporated into molecular surveillance programs to provide accurate information about the prevalence of OXA-181. A loop-mediated isothermal amplification (LAMP)-OXA48 assay overall performed well for detecting OXA-48-like enzymes but showed poor specificity due to false-positive results with non-OXA carbapenemases.