Lophophora williamsii, known for mescaline synthesis, has raised legal and ethical considerations. However, L. williamsii specimens that do not contain mescaline have been recently identified, necessitating the development of techniques to differentiate between mescaline-positive and mescaline-negative groups. Genetic markers have been explored to differentiate these specimens, complementing gas chromatography/mass spectrometry analysis. Here, we used a single-molecule real-time polymerase chain reaction to generate a full-length L. williamsii transcriptome and elucidate the mechanisms underlying mescaline biosynthesis. In total, 2,839,819 base pairs of highly accurate long reads were obtained using PacBio Iso-Seq and 70,945 unigenes were obtained through transcriptome sequencing. Known genes involved in the mescaline biosynthetic pathway were confirmed in the L. williamsii transcriptome, including 6 genes encoding tyrosine decarboxylases, 1 encoding tyrosine/DOPA decarboxylase, 215 encoding O-methyltransferases, and 129 encoding hydroxylases. Gene Ontology analysis revealed 2903 biological processes, 695 cellular components, and 1766 molecular functions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed two biosynthetic pathways, namely, phenylpropanoid and isoquinoline biosynthesis, associated with mescaline biosynthesis. To investigate genetic differences based on the presence of mescaline, Illumina Nova sequencing was conducted. The expression levels of genes involved in mescaline biosynthesis were compared to establish a candidate gene pool based on the presence of mescaline. Subsequently, functional annotation was performed on the identified candidate genes using a genetic database derived from PacBio sequencing. These findings underscore the need for a re-evaluation of forensic methods and legal regulations concerning newly identified L. williamsii specimens.