Fluoroquinolone (FQ)-resistance in Clostridium difficile has been associated with mutations in the quinolone-resistance determining region (QRDR) of gyr genes. In particular, the majority of resistant clinical isolates show mutations in codon 82 of gyrA or in codon 426 of gyrB. A real-time PCR method was developed to identify these mutations in FQ-resistant C. difficile strains. Twenty-one clinical isolates, selected as representative of the different gyr alleles known up to date, and 20 clinical isolates with unknown behavior towards FQs were used to validate the method. Each mutation was detected by real-time amplification followed by hybridization with two fluorescent probes designed with the sequence complementary to the wild-type sequences of gyr genes. The melting peak analysis of the probe-PCR product hybrid was performed on a LightCycler (Roche Diagnostic). Single and multiplex assays were performed with the same reaction conditions. In both cases, isolates showing mutations in gyr sequences had a well distinguished T(m) compared to that of isolates showing wild-type genes or silent mutated codons in the nucleotide region covered by probes. The results obtained indicate that this real-time PCR assay is a rapid, reproducible and accurate screening method of the predominant mutations determining FQ-resistance in C. difficile strains.