Objective: This study aimed to clarify the variation law of phenotypic traits such as the stem, leaf, flower, main branch ear and seed of Prunella vulgaris Linnaeus. (P. vulgaris), providing provide basic data support for protecting its wild germplasm resources. The phenotypic traits of stems, leaves, flowers, main branch ears and seeds of different wild populations of P. vulgaris were measured to reveal the phenotypic variation among and within populations of P. vulgaris, and to provide a scientific basis for the protection, utilization and collection of wild P. vulgaris germplasm resources. Methods: Variance analysis, coefficient of variation analysis, multiple comparison analysis, cluster analysis and principal component analysis were used to study the phenotypic traits of nine wild P. vulgaris populations in different regions of China. Results: The average phenotypic differentiation coefficient of nine traits was 52.04%. Inter-population variation was the main source of phenotypic variation of P. vulgaris. The average variation coefficient of each trait in the population was 21.25%, and the variation range was 15.02–28.94%. Pearson correlation analysis showed that there was a strong autocorrelation between phenotypic traits of wild P. vulgaris, and spikes and stems were greatly affected by environmental factors. According to the diameter of the stem and ear, nine wild P. vulgaris from different geographical populations could be divided into three groups. Group III (AHHS, JXFY) had the best phenotypic traits, and the traits of stem height, stem diameter, leaf length, leaf width and main branch ear length were the largest. The contribution rate of the three principal components screened by principal component analysis was 85.78%. Conclusions: We believe that the phenotypic differentiation among wild populations of P. vulgaris is at a medium level, and the variation within the population is smaller than that between populations, which may be an important reason for the sharp decline in the wild resources of P. vulgaris.