Objective To discover novel transcripts from RNA-seq data of rainbow trout under heat stress andoptimize the annotated gene structure. Methods Total RNA was extracted from the liver of rainbow trout and used toconstruct a cDNA library that was sequenced by the Illumina two-terminal sequencing Hiseq 2500 platform. Sequencing datawere assembled by Cufflinks software and aligned with the rainbow trout genome. Results Among 6555 new transcripts, 30were differentially expressed under heat stress ( P < 0. 05). A total of 3097 novel transcripts were annotated and blasted bythe GO database. There were 3617 new transcripts annotated in 284 metabolic pathways by KEGG database. The structure of19 424 annotated genes was optimized, and the 5′-ends of 14 719 genes and 3′-ends of 14 796 genes were extended.Conclusions Discovery and analysis of 6555 novel transcripts and 19 424 gene structure optimization provide a powerfulreference optimizing the genome annotation information of rainbow trout, and a more powerful theoretical basis for further understanding the mechanism of heat stress in rainbow trout.