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<i>Pueraria lobata</i> is a traditional Chinese herb in which an isoflavone <i>C</i>-glucoside, namely puerarin, has received the utmost interest due to its medicinal properties. To date, the biogenesis of puerarin, especially its <i>C</i>-glucosyl reaction in the pathway, remains poorly understood. Moreover, the transcription factors (TFs) that regulate puerarin biosynthesis in <i>P. lobata</i> have not been reported. Here, we performed phytochemical studies on the different developmental stages of the root, stem, and leaf tissues of two <i>P. lobata</i> cultivars, which suggested that both the roots and stems of <i>P. lobata</i> were the sites of puerarin biosynthesis. RNA-sequencing was conducted with the root and stem tissues of the two cultivars under different stages, and the clean reads were mapped to the recently published genome of <i>P. lobata</i> var. thomsonii, yielding the transcriptome dataset. A detailed analysis of the gene expression data, gene coexpression network, and phylogeny proposed several <i>C</i>-GTs that likely participate in puerarin biosynthesis. The first genome-wide analysis of the whole MYB superfamily in <i>P. lobata</i> presented here identified a total of 123 nonredundant <i>PlMYB</i> genes that were significantly expressed in the analyzed tissues. The phylogenetic analysis of PlMYBs with other plant MYB proteins revealed strong PlMYB candidates that may regulate the biosynthesis of isoflavones, such as puerarin.