Rheum tanguticum (Maxim. ex Regel) Maxim. ex Balf. is a herbaceous perennial plant indigenous to China, and its root and rhizomes were usually used as an important traditional Chinese medicine. However, the genomic resources are still scarce for R. tanguticum and even for Rheum genus. Transcriptome datasets from different tissues of R. tanguticum were obtained to screen the genes related to anthraquinones biosynthesis, and five free anthraquinones were also determined. Nine cDNA libraries of roots, stems and leaves were generated, and a total of 272 million high-quality reads were assembled into 257,942 unigenes. Based on the functional annotation, A total of 227 candidate enzyme genes involved in the MVA, MEP, shikimate and polyketide pathways were identified, and several differentially expressed genes found functionally associated with anthraquinones biosynthesis showed distinct tissue-specific expression patterns. Especially, we found that the expression levels of PKS III genes might result in the content differences of free anthraquinones in different tissues of R. tanguticum. Besides, 137,400 SSR loci were identified, and 64,081 SSR primer pairs were successfully designed based on these loci. Our results not only provide cues for the genetic mechanism of anthraquinone content differences in different tissues of R. tanguticum, but also lay genomic foundation for the subsequent genetic engineering and breeding for Rheum species.