Two genes, TK1280 and TK2287, encode orthologous transcription factor B proteins (TFB1 and TFB2, respectively) in the hyperthermophilic archaeon Thermococcus kodakarensis. The functional difference between their TFBs remains unknown. While TFB1 and TFB2 displayed equivalent thermostability, mRNA levels of tfb1 at 93 °C were eightfold higher than those at 60 or 85 °C, and were 4- to 10-fold greater than those of tfb2 at all temperatures. This suggests that TFB1 is the abundant TFB in T. kodakarensis and is heat-inducible. By contrast, the mRNA level of tfb2 increased at 93 °C, but the levels were less than twofold of those at 60 or 85 °C. No significant differences in growth were observed among the DTF1 (∆tfb1, ∆pyrF), DTF2 (∆tfb2 ∆pyrF), and parental host strain KU216 (∆pyrF) at 60 °C. However, DTF2 showed a decrease in cell yield at 85 °C, and both DTF1 and DTF2 showed growth defects at 93 °C. Comparative transcriptome analysis between KU216 and DTF1 or DTF2 indicated that TFB1 apparently controls the expression of genes essential for motility/adhesion, whereas TFB2 regulates genes involved in mevalonate/lipid biosynthesis. In DTF1, the ratio of cells with flagella decreased at 85 and 93 °C, and reporter studies indicated that flaB1 transcription is dependent on TFB1 at 85 °C but not at 60 °C.