Abstract: Two transcripts of Trichoderma harzianum Th-33 at the early and middle-stage of chlamydospore formation were sequenced by using high-throughput transcriptome sequencing. De novo assembly yielded 12186 unigenes with an average length of 1483 bp. A total of 6042 genes were assigned to gene ontology (GO) and 5151 unigenes mapped to 239 KEGG pathways by searching in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). A total of 6329 differentially expressed genes (DEGs) were found between the two transcripts, of which 3602 genes were up-regulated and 2727 genes were down-regulated in expression. There were 16 chitinase genes, of which 13 were up-regulated and 3 were down-regulated and 4 chitin synthase genes were all up-regulated. Meanwhile, there were 21 glucanase genes, of which 11 genes were up-regulated and 10 genes were down-regulated. KEGG enrichment analysis showed that 37 genes were significantly enriched in two KEGG pathways which were amino sugar and nucleotide sugar metabolism pathway and starch and sugar metabolism pathway. Twenty-three differentially expressed genes were found in amino sugar and nucleotide sugar metabolism pathway, and 14 differentially expressed genes in starch and sugar metabolism pathway. It was speculated that these two metabolic pathways together with the differentially expressed genes found in these two metabolic pathways were likely to be associated with the chlamydospore formation. The discovery of these metabolic pathways and genes laid a foundation for further studying the mechanism of chlamydospore formation of T. harzianum Th-33.