Abstract: Codon usage bias is quite different among different organisms. To elucidate the genetic codon preference in the most commonly used industrial strain Trichoderma reesei, codon usage analysis was performed with the opening reading frames of 9352 genes in T. reesei. The results showed that the GC content of 97% of T. reesei genes was 50%~68%, and the average content of GC3 was 70.4%. Neutral analysis and ENC-plot analysis demonstrated that the usage bias of T. reesei codons was mainly affected by selection pressure. Correlation analysis results revealed that the GC content of the genome was significantly correlated with GC1, GC2 and GC3 (P<0.05), and the number of effective codons was significantly correlated with GC3 (P<0.05). In addition, among the 24 codons frequently used by T. reesei, 22 of them contained G or C in their wobble position. Moreover, 21 codons associated with relatively effective gene expression and 4 codons relevant to optimal gene expression were further identified. The analysis also showed that the codon usage bias in T. reesei was similar to that in Trichoderma longibrachiatum and Neurospora crassa, while it was relatively different from that in Saccharomyces cerevisiae. The study herein would provide a feasible and advantageous method for genetic codon optimization in T. reesei, which would contribute to developing T. reesei as an excellent host for recombinant gene expression, as well as chassis for synthetic biology.