BackgroundPueraria is a dry root commonly used in Traditional Chinese Medicine or as food and fodder, and tuberous root expansion is an important agronomic characteristic that influences its yield. However, no specific genes regulating tuberous root expansion in Pueraria have been identified. Therefore, we aimed to explore the expansion mechanism of Pueraria at six developmental stages (P1–P6), by profiling the tuberous roots of an annual local variety “Gange No.1” harvested at 105, 135, 165, 195, 225, and 255 days after transplanting.ResultsObservations of the tuberous root phenotype and cell microstructural morphology revealed that the P3 stage was a critical boundary point in the expansion process, which was preceded by a thickening diameter and yield gain rapidly of the tuberous roots, and followed by longitudinal elongation at both ends. A total of 17,441 differentially expressed genes (DEGs) were identified by comparing the P1 stage (unexpanded) against the P2–P6 stages (expanded) using transcriptome sequencing; 386 differential genes were shared across the six developmental stages. KEGG pathway enrichment analysis showed that the DEGs shared by P1 and P2–P6 stages were mainly involved in pathways related to the “cell wall and cell cycle”, “plant hormone signal transduction”, “sucrose and starch metabolism”, and “transcription factor (TF)”. The finding is consistent with the physiological data collected on changes in sugar, starch, and hormone contents. In addition, TFs including bHLHs, AP2s, ERFs, MYBs, WRKYs, and bZIPs were involved in cell differentiation, division, and expansion, which may relate to tuberous root expansion. The combination of KEGG and trend analyses revealed six essential candidate genes involved in tuberous root expansion; of them, CDC48, ARF, and EXP genes were significantly upregulated during tuberous root expansion while INV, EXT, and XTH genes were significantly downregulated.ConclusionOur findings provide new insights into the complex mechanisms of tuberous root expansion in Pueraria and candidate target genes, which can aid in increasing Pueraria yield.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04303-x.