High-throughput sequencing technologies, often called next-generation sequencing, have revolutionized genomic research. These cutting-edge technologies are not only used for static genomes but have also been applied to the dynamic transcriptome, leading to the development of RNA sequencing (RNA-seq). RNA-seq overcomes many limitations of traditional transcriptomic methods, such as microarrays and tag-based sequencing. Despite being relatively new, RNA-seq has significantly altered our understanding of eukaryotic transcriptomes. In studying teleost fishes, both model and nonmodel species have made remarkable progress through RNA-seq in recent years. This method has contributed to the mapping and annotation of fish transcriptomes, thereby enhancing our understanding of various biological processes in fish, including development, adaptive evolution, immune responses, and stress reactions. In this chapter, we provide an overview of the entire RNA-seq process, from library construction to bioinformatics data analysis, and summarize recent biological discoveries across different fish species derived from RNA-seq studies.