Next‐generation sequencing (NGS) technologies, from the second‐generation sequencing platforms of 454/Roche, SOLiD/ABI (supported oligonucleotide ligation and detection/Applied Biosystems), and Solexa/Illumina to the transitioning system of Ion Torrent and to the third‐generation sequencing technologies of Helicos, PacBio, and Nanopore systems, have led the way in revolutionizing transcriptomics by providing unprecedented opportunities for high throughput transcriptomic research on medicinal plants, for most of which the genome/transcriptome data are not available. Transcriptome sequencing, analysis on the transcribed portion of the genome, has been used for applications ranging from gene expression profiling, protein‐coding gene annotation, and rearrangement detection to noncoding RNA discovery and genetic marker identification. This chapter discusses the characterization and application of NGS technologies in plant transcriptomic research and examples in the procedures of transcriptome analysis using 454 sequencing technology.