The presence of plasmid-encoded trimethoprim resistant dfr genes is the most common mechanism responsible for the acquisition of trimethoprim resistance. The usual method to detect the presence of these genes is hybridization with specific probes. We describe an alternative, faster and easier method, based on PCR amplification and RFLP analysis, to discriminate up to sixteen different dfr genes. © 2003 Elsevier Inc. All rights reserved.