ObjectivesDespite clinical relevance, commercially available molecular tools for accurate β-lactamase detection are limited. In this study, we evaluated the performance of the ARM-D® β-lactamase Kit, a commercially available multiplex PCR assay designed to detect nine β-lactamase genes, including the five major plasmid-mediated carbapenemases, ESBL, or AmpC genes circulating in the United States. MethodsA diverse collection of 113 Gram-negative isolates, including 42 with multiple β-lactamases was selected from the U.S. Centers for Disease Control and Prevention (CDC) & Food and Drug Administration (FDA) Antibiotic Resistance Isolate Bank, to represent the most frequently detected bacterial species carrying plasmid-mediated β-lactam resistance genes. ResultsResults were compared with whole genome sequence data. Of 164 β-lactamase gene targets with 49 unique variants, all were detected correctly without any cross-reactivity. The sensitivity and specificity were 100% (164/164) and 99.9% (852/853), respectively. ConclusionThe ARM-D® β-lactamase Kit detected a wide range of β-lactamase genotypes at a low upfront cost. The Streck assay represents a suitable, comprehensive tool for the detection of key β-lactamase resistance genes of public health concern in the United States.