Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23S rRNA gene inteal transcribed spacer sequence for the genotyping of mycobacteria to the species level. A pair of primers and a set of genus- and species-specific probes were designed from the conserved and polymorphic regions of the 16S rRNA gene, inteal transcribed spacer, and 23S rRNA gene sequences of mycobacteria. We used a novel multiplex probe array for identification of 266 clinical specimens obtained from patients with mycobaterial infection. The results showed that the overall specificity and sensitivity of our novel probe array were both 100% for the genus-specific probe and Mycobacterium tuberculosis complex-specific probe. There were 79.3% (23/29) of nontuberculous mycobacteria which could be identified to the species level directly in the specimens from China. Some intraspecies heterogeneity in M. avium, M. intracellulare, M. chelonae and M. abscessus was observed. With the increase of sequences of inteal transcribed spacer and numbers of whole microbial genomes, and further optimization of probes, the multiplex probe array will become a promising tool for the rapid and accurate identification of mycobacteria in ordinary clinical laboratories.