Reverse transcription differential display PCR (RT-DD-PCR) was used to identify genes that are specifically expressed by Metarhizium anisopliae when it contacts the host insect cuticle. Using a homology-based subtilisin-like protease primer we identified a hitherto unsuspected differentially expressed subtilisin-like protease (Pr1B) encoding gene. The deduced amino acid sequence shows 54% similarity to the well characterized Pr1A subtilisin of M. anisopliae and karyotype analysis revealed that Pr1A and Pr1B are located on separate chromosomes. Like Pr1A, Pr1B is synthesized as a large precursor (1158 nucleotides; deduced molecular mass = 40031 Da) containing a signal peptide, a propeptide and the mature protease (283 aa; deduced molecular mass = 28714 Da). However, Pr1B possesses several substitutions in the highly conserved sequences comprising the active sites of subtilisins. In particular, the substitution of Thr220 by serine is unique to Pr1B. Substitution of Asn155 by glycine is also very unusual, and we discuss the likely effects these changes will have on the catalytic efficiency of Pr1B.