Trichoderma virens, an imperfect fungus, is used as a biocontrol agent to suppress plant disease caused by soilborne fungal pathogens. Antimicrobial peptides it produces include peptaibols of 11, 14, and 18 amino acids in length. These peptaibols were previously reported to be synthesized by a non-ribosomal peptide synthetase (NRPS) encoded by the Tex1 gene in strain Tv29-8. The present study examined the Tex1 homolog in a commercially relevant T. virens strain, G20. Although the gene in G20 was 99% identical in DNA sequence to Tex1 in the 15.8 kb compared, gene disruption results indicate that it is only responsible for the production of an 18-mer peptaibol, and not 11-mer and 14-mer peptaibols. Additional NRPS adenylate domains were identified in T. virens and one was found to be part of a 5-module NRPS gene. Although the multimodule gene is not needed for peptaibol synthesis, sequence comparisons suggest that two of the individual adenylate domain clones might be part of a separate peptaibol synthesis NRPS gene. The results indicate a significant diversity of NRPS genes in T. virens that is unexpected from the literature.