Due to excessive human disturbance, the germplasm resources of Quasipaa spinosa are experiencing a decline, and the gradual reduction in genetic diversity has become an urgent issue that needs to be addressed. The objective of this study was to identify the germplasm of different geographical populations, comprehensively evaluate their characteristics, and identify excellent breeding populations. This would provide important reference materials for subsequent population selection and the optimization of germplasm resources. The study employed methods such as single-factor analysis of variance, principal component analysis, cluster analysis, and discriminant analysis to conduct morphological analyses of 120 cultured individuals from four geographical populations (Guixi YJFGX, Yifeng LHSYF, Yudu LHSYD, and Taining LHSTN). Genetic differences among Quasipaa spinosa populations were also analyzed based on simplified genome sequencing results. The findings revealed variations in the morphological characteristics among the four geographical populations. Particularly notable were the differences between LHSYD and the other three populations; however, there were similarities in morphological traits between LHSTN and YJFGX, without significant differentiation. Four geographical populations exhibit a relatively common sexual dimorphism in body limb length and other related indicators. Based on morphological characteristics, the four populations can be roughly divided into three major branches: the LHSTN population and the YJFGX population each form a branch, while the LHSYF population and the LHSYD population comprise another branch. The observed heterozygosity (H_o) ranged from 0.035 to 0.038, lower than the expected heterozygosity (H_e) ranging from 0.103 to 0.129. Moreover, the average inbreeding coefficient ranged from 0.197 to 0.287, indicating a high level of inbreeding within these populations. A relatively high degree of genetic differentiation was observed between LHSTN and LHSYF (F_st=0.303), LHSYD (F_st=0.277), and YJFGX (F_st=0.276) populations; however, only moderate genetic differentiation existed among LHSYF, LHSYD, and YJFGX populations. In summary, (1) there is morphological variation among the four studied populations, but not at the subspecies level and (2) the overall genetic diversity of the population was relatively low, indicating a high degree of genetic differentiation and independent genetic information. This characteristic makes them suitable as parental sources for crossbreeding, thereby enhancing the genetic diversity of the local population and optimizing germplasm resources.