The SigB concentrations in clinical isolates of Staphylococcus aureus were measured to examine their correlation with the antibiotic resistance. The SigB concentrations in methicillin-resistant S. aureus (MRSA) were higher than in the control strain, N315, and many of methicillin-susceptible S. aureus (MSSA). Sequencing analyses of the sigB genes revealed that the strains exhibiting the high SigB concentrations have three amino acid substitutions in SigB: I11V, D141N, and Q256K. Further analysis using isogenic mutants demonstrated that D141N (or both D141N and Q256K) is essential to maintain the high SigB concentration. These substitutions affected the UV tolerance, but had no effect on the antibiotic resistance. The SigB activity was affected by these substitutions toward the stationary phase, but not during the transient heat shock response.