OBJECTIVES: qnr genes are plasmid-mediated quinolone resistance genes. Five qnr families have been described with several alleles (7 alleles of qnrA, 53 alleles of qnrB, 1 allele of qnrC, 1 allele of qnrD and 6 alleles of qnrS). Their detection requires a PCR specific for each qnr family and further sequencing for allele characterization. METHODS: High-resolution melt curve analysis (HRMA) was coupled to multiplex and simplex real-time PCR assays for detection and characterization of qnrA, qnrB and qnrS alleles. The protocol was set using 27 reference strains harbouring the most frequent alleles and was applied to 55 clinical isolates unknown for qnr positivity. RESULTS: Out of the 27 reference strains tested, 21 alleles showed distinct profiles using HRMA: 6 qnrA, 12 qnrB and 3 qnrS. For the qnrB alleles showing similar profiles, we gathered them into four groups that were easily distinguished. For the alleles that we could not test, in silico analysis showed that they would be identified using the HRMA protocol set. Among the clinical isolates, 28 qnr-positive isolates were detected and the qnr allele was characterized as 8 qnrA1, 4 qnrB1, 5 qnrB2, 3 qnrB4, 1 qnrB8, 1 qnrB5, 3 qnrS1 and 1 qnrS2, with concordant results with PCR sequencing. Two new qnrB alleles were detected and distinguished using HMRA. They were further designated as qnrB25 and qnrB42. CONCLUSIONS: We developed an HRMA assay for characterizing the qnr alleles in clinical isolates. This high-throughput method can be used to screen a large number of isolates. This method allowed the detection of new qnrB alleles.