The salt-tolerant Torulopsis versatilis mutant strain, T3-5, is able to grow in YPD medium containing 22% salt, whereas the original strain, T, used for soy sauce fermentation was only able to grow in the presence of 20% or less. Biochemical analyses showed that strain T3-5 had a 22% higher intracellular glycerol level than T. Sequencing of the glycerol transporter gene FPS1 revealed a change from G to A at position 198 upstream of the start codon, changes from G to A at positions 93 and 1084, respectively, within the FPS1 gene, and insertions of T base at positions 285 and 310, respectively, downstream of the termination codon when comparing the corresponding sequences of strains T and T3-5. An inverse relationship between osmotic pressure and the FPS1 transcription level was observed for both T and T3-5 using qPCR. Therefore, it is possible that lower transcription levels of FPS1 are at least partly responsible for increased osmotolerance in strain T3-5. Overexpression of T and T3-5 FPS1 in Saccharomyces cerevisiae resulted in lower salt tolerance, especially in the presence of 12% salt. The data suggest that decreased expression levels of and/or alteration in the FPS1 protein contributed to increased salt tolerance of T3-5.