Rheum tanguticum is a perennial herb and an important medicinal plant, with anthraquinones as its main bioactive compounds. However, the specific pathway of anthraquinone biosynthesis in rhubarb is still unclear. The accumulation of anthraquinones in different tissues (root, leaf, stem and seed) of R. tanguticum revealed considerable variation, suggesting possible differences in metabolite biosynthetic pathways and accumulation among various tissues. To better illustrate the biosynthetic pathway of anthraquinones, we assembled transcriptome sequences from the root, leaf, stem and seed tissues yielding 157,564 transcripts and 88,142 unigenes. Putative functions could be assigned to 56,911 unigenes (64.57%) based on BLAST searches against annotation databases, including GO, KEGG, Swiss-Prot, NR, and Pfam. In addition, putative genes involved in the biosynthetic pathway of anthraquinone were identified. The expression profiles of nine unigenes involved in anthraquinone biosynthesis were verified in different tissues of R. tanguticum by qRT-PCR. Various transcription factors, including bHLH, MYB_related, and C2H2, were identified by searching unigenes against plantTFDB. This is the first transcriptome analysis of different tissues of R. tanguticum and can be utilized to describe the genes involved in the biosynthetic pathway of anthraquiones, understanding the molecular mechanism of active compounds in R. tanguticum.