Abstract: ObjectiveTo investigate the optimal pretreatment method for the identification of Nocardia using matrix assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS). MethodsAtotal of 93 clinical isolates of Nocardia were identified by MALDI-TOF MS using three pretreatment methods: diformic acid method, glass bead oscilation-formic acid-acetonitrile extraction, and in-situ formic acid extraction. The results were evaluated with gene sequencing results,including 16S rRNA and secAl gene sequencing as the reference method. Statisticalagreement was assessedusingKappa statistics. ResultsAmong the 93Nocardia strains，91 (97.8%)were successfully identified at the species level using the diformic acid method, 88(94.6%）using the glass bead oscillation-formic acid-acetonitrile extraction method,and 64 (68.8%）using the in-situ formic acid extraction method. Compared with genesequencingtKappavaluesfospecies-lvlidentifcationcapabilitwere.andrespectivelyFortheNocardia abscessus complex，the species-level identification accuracies of the three methods were 95.5%(21/22)，86.4%(19/22)，and 36.4% (8/22)，respectively，with corresponding Kappa values of O.900，0.730and 0.251. ConclusionThe diformic acid method is simple, rapid, and highly accurate. For the Nocardia abscessus complex, this method demonstrated higher identification accuracy than the glass bead oscillation-formic acid-acetonitrile extraction method. It is recommended to use the diformic acid method for pretreatment in MALDI-TOF MS identification of Nocardia.