Abstract: Objective To evaluate the consistency of mycobacteria identification by the matrix -assisted laser desorption/ ionization time-of- flight mass spectrometry ( MALDI-TOF MS) and multilocus sequencing, and then provide a theoretical basis for the clinical application of MALDI-TOF MS ( BioMerieux,IVD V3.0) in identifying mycobacteria and further understand the local distribution of mycobacteria. Methods The RNA polymerase β subunit ( rpoB)，heat shock protein 65 ( hsp65) and 16S rRNA partial sequences of 751 strains of mycobacteria identified by MALDI-TOF MS in Weifang No. 2 People' s Hospital during July 2016 and October 2017 were identified by the multilocus sequencing. The results of joint identification of the three genes were used as the gold standard to evaluate the accuracy of mycobacteria identification by MALDI-TOF MS. Meanwhile, the distribution of 6 350 strains of mycobacteria isolated from clinical samples in the past 5 years and identified by MALDI-TOF MS was analyzed. Results Referring to the sequencing results of rpoB, hsp65 and 16S rRNA, the identification accuracies of MALDI-TOF MS were 99.66% for M. tuberculosis complex and 97.40% for non-tuberculosis mycobacteria ( NTM)，respectively. In the past 5 years, 15.51% of clinically isolated mycobacteria were NTM in W eifang region, and the main NTM species were slow-growing mycobacteria ( 90.46%)，of which M. intracellulare accounted for the highest proportion (73.20% )，followed by M. kansasii ( 12.08% ). Most of the fast-growing mycobacteria were M. abscessus (6.19%).Conclusion MALDI-TOF MS is reliable in the identification of mycobacteria and has important clinical value. The NTM in Weifang region is dominated by M. intracellulare.