Technical Abstract: Investigation into the molecular mechanisms of antimicrobial resistance has been confounded by the emergence of multi drug resistant bacteria. Characterization of these strains can require hundreds of assays to identify the genes conferring antibiotic resistance. We have designed a microarray for the rapid detection of antibiotic resistance genes found in diverse bacteria. The array was designed to detect over 90 genes commonly found in a variety of bacteria which confer resistance to an assortment of antimicrobial drug classes. Oligonucleotide probes were designed, synthesized, robotically arrayed onto glass slides which were processed, hybridized, scanned and scored following standard microarray techniques. Preliminary tests were conducted with diverse collections of Gram negative and Gram positive pathogens and commensal bacteria. The probes demonstrated high specificity, low background and low cross reactivity. Test strains were found to harbor antimicrobial resistance genes consistent with their antibiograms. Several genes were confirmed by alternate methods including PCR and Southern blotting. Additionally, conjugation experiments established that the microarray could be used to trace the horizontal transfer of multiple antibiotic resistance genes. The results demonstrate that these techniques can be used to quickly design a microarray for detecting horizontally exchanged DNA elements in virtually any bacteria.