Pinellia (Pinellia ternata (Thunb.) Breit.), as important medicinal plant, has been used to treat various ailments for a long time. The sixteen ploid plant (2n = 16 * 13 = 208) Pinellia T2Plus line was obtained from an octoploid (2n = 8 * 13 = 104) T2 line by chromosome-doubling technique. Compared with T2 line, the content of various medicinal components (polysaccharide, guanosine, adenosine and ephedrine) was increased in T2Plus line. In this study, the transcriptome of T2 line and T2Plus line were characterized by RNA sequencing (RNA-seq) technology. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis on differential expressed unigenes (DEGs) revealed that multiple metabolic pathway were enriched significantly, such as 'Starch and sucrose metabolism', 'Purine metabolism', 'Photosynthesis' and six transcription factors (MYB, WRKY, bHLH, lateral organ boundaries domain (LBD), homeodomain-zipper (HD-ZIP) and Ethylene-responsive factor (ERF)) play a key role in difference of transcriptome between T2 line and T2Plus line. These metabolic pathways and transcription factors may play an important role in the difference of medicinal components and epigenetic features between these two Pinellia cultivars. This conclusion provides a robust theoretical basis for the mechanism of the formation of medicinal ingredients in Pinellia cultivars.