Poison hemlock (Conium maculatum L.) is a notorious weed containing the potent alkaloid coniine. Only some of the enzymes in the coniine biosynthesis have so far been characterized. Here, we utilize the next-generation RNA sequencing approach to report the first-ever transcriptome sequencing of five organs of poison hemlock: developing fruit, flower, root, leaf, and stem. Using a de novo assembly approach, we derived a transcriptome assembly containing 123,240 transcripts. The assembly is deemed high quality, representing over 88% of the near-universal ortholog genes of the Eudicots clade. Nearly 80% of the transcripts were functionally annotated using a combination of three approaches. The current study focuses on describing the coniine pathway by identifying in silico transcript candidates for polyketide reductase, l-alanine:5-keto-octanal aminotransferase, γ-coniceine reductase, and S-adenosyl-l-methionine:coniine methyltransferase. In vitro testing will be needed to confirm the assigned functions of the selected candidates.