Background <p>Radix Bupleuri, derived from the dried roots of <i>Bupleurum chinense</i> DC., is a well-documented phytomedicine in global pharmacopoeias and a common constituent in herbal formulations. While previous studies have hinted at regional variations in the chemical composition of <i>B. chinense</i>, a comprehensive understanding of its morphological, genetic, and chemical diversity across China remains incomplete.</p> Objective <p>This study aims to investigate the infraspecific variation of <i>B. chinense</i> by analyzing its morphological, genetic, and chemical phenotypes.</p> Methods <p>Wild <i>B. chinense</i> specimens were collected from 31 locations spanning nine Chinese provinces/municipalities, representing a wide range of its natural distribution. A multi-faceted approach combining 21 morphological traits, plastid genome sequencing, and chemical analysis was employed to explore infraspecific variation and clustering patterns.</p> Results <p>Distinct infraspecific variation was revealed through integrated morphological and molecular data. Morphological clustering analysis identified two geographically associated clusters, roughly corresponding to coastal and inland regions. Although plastid genome sequencing of 40 specimens showed high sequence identity, population structure analysis detected variable hotspots. Both maximum likelihood (ML) tree and population structure results consistently identified three distinct clades, which mirrored the patterns observed in morphological clustering. Quantitative analysis of saikosaponins content in 10 representative specimens across the three clades demonstrated significant chemotype variation. Notably, samples from Anhui Province exhibited the highest saikosaponins content, while those from Shanxi Province showed the lowest levels. This chemotype variation, coupled with observed genetic diversity, suggests that <i>B. chinense</i> germplasm from Clade I (particularly from Anhui Province) represents a promising wild resource for further development.</p>