Derris fordii and Derris elliptica belong to the Derris genus of the Fabaceae family, distinguished by their high isoflavonoid content, particularly rotenoids, which hold significance in pharmaceuticals and agriculture. Rotenone, as a prominent rotenoid, has a longstanding history of use in pesticides, veterinary applications, medicine, and medical research. The accumulation of rotenoids within Derris plants adheres to species-specific and tissuespecific patterns and is also influenced by environmental factors. Current research predominantly addresses extraction techniques, pharmacological applications, and pesticide formulations, whereas investigations into the biosynthesis pathway and regulatory mechanism of rotenoids remain relatively scarce. In this study, we observed notable differences in rotenone content across the roots, stems, and leaves of D. fordii, as well as within the roots of D. elliptica. Utilizing RNA sequencing (RNA-seq), we analyzed the transcriptomes and expression profiles of unigenes from these four tissues, identifying a total of 121,576 unigenes. Differentially expressed genes (DEGs) across four comparison groups demonstrated significant enrichment in the phenylpropanoid and flavonoid biosynthesis pathways. Key unigenes implicated in the rotenoid biosynthesis pathway were identified, with PAL, C4H, CHS, CHI, IFS, and HI4OMT playing critical roles in D. fordii, while IFS and HI4OMT were determined to be essential for rotenoid biosynthesis in D. elliptica. These findings enhance our understanding of the biosynthesis mechanism of rotenoids in Derris species. The unigenes identified in this study represent promising candidates for future investigations aimed at validating their roles in rotenoid biosynthesis.