Gentiana straminea is a popular Tibetan medicine that has been used for thousands of years in China to treat various diseases and conditions. Although it has multiple pharmaceutical purposes and important economic plant resource in China, transcriptome and molecular base still known limited. In flowering season, samples were collected from different tissues, using the NGS Illumina. Solexa platform, about 58.85 million sequencing reads were generated and assembled de novo, yielding 78,764 high quality unigenes with an average length of 1090bp. Gene Ontology (GO), KEGG pathway mapping showed that 49,033 of these were identified as putative homologs of annotated sequences in the protein databases. Among them, candidate genes associated with iridoid, flavonoid and anthocyanin were identified. Further the key enzymes involved to iridoid and flavonoid synthesis pathway were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) on different tissues, the flower and root had the higher expression than leaves. In addition, 7591 SSR markers were identified from the unigenes of the G. straminea transcriptome. The foundation of G. straminea provided the important resource for facilitating to study molecular and functional genomics of it and related this species on the Qinghai-Tibet Plateau.