In this study, a multisite SNP genotyping and macrolide (ML) susceptibility gene test method for () was developed based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The detection limit of this method for nucleic acids was 10 -10 copies/reaction. Six SNP site-based genotyping and 3 ML susceptibility sites could be detected simultaneously based on multiplex PCR and mass probe. Using the method constructed in this study, 141 Chinese clinical isolates were divided into 8 SNP types. All the SNP test results for the ML susceptibility gene were in line with those of the 23S rRNA sequencing results. With this method, the multisite SNP genotyping and ML susceptibility determination of can be completed simultaneously in one test, which greatly reduces the workload and cost, improves the genotyping ability of and deserves clinical application.