Alisma orientale
(Sam.) Juzep (
A
.
orientale
) is an important medicinal plant in traditional Chinese medicine. In this study,
de novo
RNA-seq of
A
.
orientale
was performed based on the cDNA libraries from four different tissues, roots, leaves, scapes and inflorescences. A total of 41,685 unigenes were assembled, 25,024 unigene functional annotations were obtained by searching against the five public sequence databases, and 3,411 simple sequence repeats in
A
.
orientale
were reported for the first time. 15,402 differentially expressed genes were analysed. The morphological characteristics showed that compared to the other tissues, the leaves had more chlorophyll, the scapes had more vascular bundles, and the inflorescences contained more starch granules and protein. In addition, the metabolic profiles of eight kinds of alisols metabolite profiling, which were measured by ultra-Performance liquid chromatography-triple quadrupole-mass spectrometry showed that alisol B 23-acetate and alisol B were the major components of the four tissues at amounts of 0.068~0.350 mg/g and 0.046~0.587 mg/g, respectively. In addition, qRT-PCR validated that farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-CoA reductase should be considered the critical candidate genes involved in alisol biosynthesis. These transcriptome and metabolic profiles of
A
.
orientale
may help clarify the molecular mechanisms underlying the medicinal characteristics of
A
.
orientale
.