Aims:  To isolate, characterize, and identify lactic acid bacteria (LAB) in suan‐tsai (fermented mustard), a traditional fermented food in Taiwan. Methods and Results:  Suan‐tsai samples were collected at five time points from a fixed fermenting bucket. Fifty cultures were isolated from suan‐tsai samples, and isolates were divided into classes by phenotype and then into groups by restriction‐fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Phenotypic and biochemical characteristics identified two different bacterial groups (A and B), and the results showed that Pediococcus pentosaceus was the most abundant LAB during the initial fermentation time. However, the more NaCl‐tolerant species Tetragenococcus halophilus took the place of P. pentosaceus and became the most abundant LAB later. All isolates were grown in de Man, Rogosa, and Sharpe (MRS) broth containing 6% NaCl, but T. halophilus could grow only in MRS broth containing 10% NaCl. Conclusions:  These results suggest that the LAB P. pentosaceus and T. halophilus play roles in the fermentation of suan‐tsai. Significance and Impact of the Study:  This is the first report describing the distribution and varieties of LAB that exist in the suan‐tsai fermentation process.