Invasive fungal infections are associated with high mortality and morbidity rates, especially in immunocompromised and critically ill patients (1-3). Although the most important causes of opportunistic mycoses are Candida species, especially Candida albicans, the incidence of invasive fungal infections due to nonalbicans Candida species is increasing, especially in China (4-8).The National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program is a nationwide, multicenter surveillance network established in July 2009 to provide updated information on the epidemiology of invasive fungal infections in China (9). The continual expansion of the CHIF-NET program has led to a dramatic increase in the number of laboratories submitting isolates. Therefore, an accurate, time-saving, cost-effective, and userfriendly yeast identification method is needed to replace the highcost and labor-intensive sequence-based methods used by the central laboratory.Several studies have reported that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an accurate, rapid, and inexpensive method for identification of clinically relevant yeasts (10, 11). In our previous study, we evaluated the performance of the Vitek MS system (bioMérieux, France) for identifying yeast isolates collected as part of the CHIF-NET program in 2011 (12). In the present study, we further systematically compared the performance of the Bruker Biotyper MS (Bruker Daltonics GmbH, Germany) and the Vitek MS systems for the identification of a large number of clinically relevant yeast isolates collected through the CHIF-NET program (2012 to 2013).(This study was presented in part at the 19th Congress of the International Society for Human and Animal Mycology (ISHAM), Melbourne, Australia, 2015.)The 2,683 yeast isolates analyzed in this study were all derived from patients with invasive candidiasis from 48 clinical microbiology laboratories of hospitals situated in 24 provinces across China during the period August 2012 to July 2013. The majority of the nonduplicate isolates were obtained from blood (44.2%), followed by ascitic fluid (18.2%), intravascular catheters (7.6%), pus (7.3%), and cerebrospinal fluid (5.9%) (see Table S1 in the supplemental material). The isolates were inoculated onto CHROMagar Candida medium (CHROMagar, Paris, France), incubated for 48 h at 35°C, and then simultaneously identified using the Vitek MS and Bruker Biotyper MS systems.For the Vitek MS v.2.0 system, proteins were extracted as recommended by the manufacturer. Briefly, a small portion of a single colony was directly spotted onto a target plate and covered with 0.5 l of formic acid (bioMérieux). All mass profiles were analyzed using the Vitek MS database (MS-ID v.2.0 knowledge base clinical use). For the Bruker Biotyper MS system, pure yeast isolates (each from a single colony), were directly smeared onto the target plate (Bruker Daltonics GmbH) and overlaid with 1 l of 70% formic acid (Sigma-Aldrich). Each spectrum was analyzed by ...