Aims: A new cellobiohydrolase (CBH) gene (60) from Chaetomium thermophilum was cloned, sequenced and expressed in Pichia pastoris. Methods and Results: Using RACE-PCR, a new thermostable CBH gene (60) was cloned from C. thermophilum. The cDNA of the CBH was 1607 bp and contained a 1356 bp open reading frame encoding a protein CBH precursor of 451 amino acid residues. The mature protein structure of C. thermophilium CBH3 only comprises a catalytic domain and lacks cellulose-binding domain and a hinge region. The gene was expressed in P. pastoris. The recombinant CBH purified was a glycoprotein with a size of about 48.0 kDa, and exhibited optimum catalytic activity at pH 5-0 and 60 degrees C. The enzyme was more resistant to high temperature. The CBH could hydrolyse microcrystalline cellulose and filter paper. Conclusions: A new thermostable CBH gene of C. thermophilum was cloned, sequenced and overexpressed in P. pastoris. Significance and Impact of the Study: This CBH offers an interesting potential in saccharification steps in both cellulose enzymatic conversion and alcohol production.