Objective To evaluate and compare yeast identification ability between Bruker Microflex matrix-assisted laser desorption ionization-time of flight mass spectrometry( MALDI-TOF MS) and Vitek 2 Compact automatic microbial analysis system.Methods Retrospective study.Totally 742 strains of yeast isolated from clinical specimens during March 2013 to March 2014 in Xinhua Hospital, Shanghai Jiao Tong University School of Medicine were identified by Bruker Microflex MALDI-TOF MS and Vitek 2 Compact automatic microbial analysis system simultaneously.The strains with discordant results were validated by gene sequencing.Results The coincidence rate of 699 Candida identified by Bruker Microflex MALDI-TOF MS or Vitek 2 Compact system was 100.0%(699/699) and 99.6%(696/699) to the species level, respectively and the coincidence rate of 43 yeast-like fungi strains identified was 90.7%(39/43) and 79.1%(34/43) to the species level, respectively.Penicillium marneffei could not be identified by both two instruments, but protein profile of Penicillium marneffei by MALDI-TOF MS was established.Conclusions The coincidence rate of yeast identified by Bruker Microflex MALDI-TOF MS is higher than that of Vitek 2 Compact system.Using Bruker Microflex MALDI-TOF MS to identify yeast especially Candida and yeast-like fungus is fast, simple, low-cost, accurate, and it can be used in routine work of ordinary yeast identification in clinical microbiology laboratory.