Background Pueraria lobata (Willd.) Ohwi is a valuable herb used in traditional Chinese medicine. Isoflavonoids are the major bioactive compounds in P. lobata , namely puerarin, daidzin, glycitin, genistin, daidzein, and glycitein, which have pharmacological properties of anti-cardiovascular, anti-hypertension, anti-inflammatory, and anti-arrhythmic. Methods To characterize the corresponding genes of the compounds in the isoflavonoid pathway, RNA sequencing (RNA-Seq) analyses of roots, stems, and leaves of P. lobata were carried out on the BGISEQ-500 sequencing platform. Results We identified 140,905 unigenes in total, of which 109,687 were annotated in public databases, after assembling the transcripts from all three tissues. Multiple genes encoding key enzymes, such as IF7GT and transcription factors, associated with isoflavonoid biosynthesis were identified and then further analyzed. Quantitative real-time PCR (qRT-PCR) results of some genes encoding key enzymes were consistent with our RNA-Seq analysis. Differentially expressed genes (DEGs) were determined by analyzing the expression profiles of roots compared with other tissues (leaves and stems). This analysis revealed numerous DEGs that were either uniquely expressed or up-regulated in the roots. Finally, quantitative analyses of isoflavonoid metabolites occurring in the three P. lobata tissue types were done via high-performance liquid-chromatography and tandem mass spectrometry methodology (HPLC-MS/MS). Our comprehensive transcriptome investigation substantially expands the genomic resources of P. lobata and provides valuable knowledge on both gene expression regulation and promising candidate genes that are involved in plant isoflavonoid pathways.