Abstract: Objective: To investigate the value of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) in S. typhimurium homology analysis. Methods: Forty-four S. typhimuium strains isolated from Guangzhou were typed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, which was compared with pulse-field gel electrophoresis(PFGE) to analyze the main patterns of mass spectrogram. Results: The PFGE results showed that 44 S. typhimuium strains could be divided into 15 types. At 80% of the clustering similarity, 44 S. typhimuium strains could be divided into 8 MALDI-TOF MS types, which was accordance to the PFGE results. The MS1(52.3%) was the main type, which contained multiple PFGE patterns. Conclusions: The classification results of MALDI-TOF MS show the relative concentration of kinship of S. typhimuium isolated from Guangzhou, which is a simple, rapid, high throughput method of classification.