Paris polyphylla Smith is a perennial medicinal plant that is wildly distributed in China, and polyphyllin is the representative medicinal ingredient from the rhizome of this plant. To date, the mechanism of steroidal saponin biosynthesis in P. polyphylla is still unclear. In this study, comparative de novo transcriptome sequencing of the rhizomes, leaves and stems from P. polyphylla were performed with the Illumina HiSeq 2500 platform. A total of 53.71 Gb of transcriptomic data was obtained from the distinct organs, and 24,297 annotated unigenes out of 50,428 assembled unigenes were achieved by aligning with the nonredundant protein sequence database; evolutionary genealogy of genes: Nonsupervised Orthologous Groups; Pfam; Swiss-Prot; euKaryotic Orthologous Groups; Gene Ontology; Kyoto Encyclopedia of Genes and Genomes (KEGG); and the Clusters of Orthologous Groups databases, and 10,141 simple sequence repeat loci were identified based on their transcriptome profiles. KEGG pathway enrichment assigned 38, 32, and 7 unigenes to terpenoid backbone biosynthesis, steroid biosynthesis, and the sesquiterpenoid and triterpenoid biosynthesis pathway, respectively. A total of 25 unigenes that encoded 17 key enzymes were identified as being involved in steroidal saponin biosynthesis in P. polyphylla. CYP90B1 (4 unigenes) and CYP724B1 (2 unigenes) from the annotated cytochrome P450 s (36 unigenes) were inferred to play probable roles in steroidal saponin biosynthesis. The phylogenetic analysis of squalene monooxygenase and cycloartenol synthase from P. polyphylla indicated close homology with multiple monocots. Furthermore, the differentially expressed genes were identified by comparison of the transcriptome profiles of distinct organs in a pairwise fashion, and the expression patterns of the candidate unigenes that were potentially responsible for steroidal saponin biosynthesis were analyzed thoroughly and coupled with the determination of polyphyllin contents in the different organs. These findings offer abundant genetic resources and a molecular basis to further study polyphyllin biosynthesis in P. polyphylla and its closely related Paris species.