Abstract: Objective：To evaluate the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification ofCorynebacterium spp. Methods：Fifty-eight clinical isolates of Corynebacterium spp. were collected. Species identification was performed by both MALDI-TOF MS and 16S rRNA gene sequencing. MALDI-TOF MS protein phylogenetic tree of different Corynebacterium species was constructed by MALDI-Biotyper software. Results：The results of identification for 54 isolates by 16S rRNA gene sequencing were coincident with those by MALDI-TOF MS, including 34 of C.straitum strains, 11 of C.jeikeium strains, 3 of C.resistens strains, 2 of C.aurimucosum strains, 2 of C.amycolatum strains and 2 of C.glucuronolyticum strains. Four isolates could not be identified into any species by 16S rRNA gene sequencing, but they were identified as C.mucifaciens (2 isolates), C.singulare (1 isolate) and C.pseudodiphtherititum (1 isolate) by MALDI-TOF MS. Conclusion：MALDI-TOF MS should be available for rapid identification of Corynebacterium spp.