Objective To investigate the variation of chemical characteristics with environmental factors and establish a relationship between morphological characters and chemical composition of Prunella vulgaris collected in different areas of China. Methods Twelve phenotypic traits and three chemical compositions were assessed in 28 populations of P. vulgaris collected from different locations in China. Results The variability ranges observed at phenotypic and chemical levels were polymorphic. According to the morphological traits, 28 populations of P. vulgaris could be grouped into six clusters, and two morpho-types could be clearly distinguished. Perceptible differences could be discerned in the plant height, leaf length, corolla length, calyx length, fruiting spikes length, and maturity period. Based on three kinds of components including ursolic acid, total flavonoids, and total polysaccharides, all populations could be identified as four types. Cluster IV showing high content of ursolic acid, total flavonoids, and total polysaccharides could be utilized to develop superior derivatives. Conclusion The variation of chemical characteristics is influenced by the genetic and environmental factors, such as soil, climate, longitude, and altitude. It provides a solid basis for efficiently evaluating qualities and establishing good agricultural practices for P. vulgaris. Objective To investigate the variation of chemical characteristics with environmental factors and establish a relationship between morphological characters and chemical composition of Prunella vulgaris collected in different areas of China. Methods Twelve phenotypic traits and three chemical compositions were assessed in 28 populations of P. vulgaris collected from Results The variability ranges observed at phenotypic and chemical levels were polymorphic. According to the morphological traits, 28 populations of P. vulgaris could be grouped into six clusters, and two morpho-types could be clearly distinguished. Perceptible differences could Based on three kinds of components including ursolic acid, total flavonoids, and total polysaccharides, all populations could be identified as four types. be discerned in the plant height, leaf length, corolla length, calyx length, fruiting spikes length, and maturity period. Cluster IV showing high content of ursolic ac id, total flavonoids, and total polysaccharides could be utilized to develop superior derivatives. Conclusion The variation of chemical characteristics is influenced by the genetic and environmental factors, such as soil, climate, longitude, and altitude. It provides a solid basis for efficient evaluating qualities and establishing good agricultural practices for P. vulgaris.