The molecular identification and its drug-resistance profile are important to choose the correct therapy. This work developed a multiplex real-time PCR (mqPCR) for detection of clarithromycin resistance genes for the group. Isolates received by Adolfo Lutz Institute from 2010 to 2012, identified by PCR restriction enzyme analysis of a fragment of the 65 gene (PRA-65) as type 1 (=135) and 2 (=71) were used. Drug susceptibility test (DST) for CLA were performed with reading on days 3 and 14. Subespecies identification by 65 and B genes sequencing and (41) and genes for mutation detection and primer design were performed. (41) gene deletion was detected by conventional PCR. Primers and probes were designed for five detections: (41) gene full size and with deletion; (41) gene T28 and C28; gene A2058. In total, 191/206 (92.7 %) isolates were concordant by all methods and 13/206 (6.3 %) were concordant only between molecular methods. Two isolates (1.0 %) were discordant by mqPCR compared to gene sequencing. The mqPCR obtained 204/206 (99.0 %) isolates in agreement with the gold standard, with sensitivity and specificity of 98 and 100 %, respectively, considering the gold standard method and 92 and 93 % regarding DST. The mqPCR developed by us proved to be an easy-to-apply tool, minimizing time, errors and contamination.