[Objective] In order to better understand the population variation of cork oak leaf anatomical structure and its relationship with environmental factors. [Methods] The leaves of 28 naturally distributed Quercus variabilis populations in China were studied by conventional paraffin sections combined with optical microscopy. Nested analysis of variance and Pearson correlation analysis were used to analyze the relationship between population variation of leaf anatomical structure and environmental factors. [Results] (1) there were significant differences in leaf anatomic traits among 28 populations, the average coefficient of variation was 7.90% ~ 14.70%, and the variation range of the same leaf anatomic traits was different among different populations. (2) There were significant differences among the 9 anatomic traits within and among populations, and the average phenotypic differentiation coefficient was 37.44%. The variation of anatomic traits mainly came from within populations. (3) Leaf thickness, epidermis thickness, palisade tissue thickness and spongy tissue thickness were significantly positively correlated with latitude, and average annual temperature and annual precipitation had significant effects on the epidermis thickness and palisade tissue thickness. [Conclusion] The results show that Q. variabilis has rich genetic, in order to adapt to low temperature and drought, the leaves of Q. variabilis showed a trend of thickening. The results can provide theoretical basis for understanding environmental adaptation strategies of Q. variabilis.