Stratification is an important phenomenon in natural forests. The mixed pineoak forests along the Nanpan River in southwest China was clearly formed by two layers in the vertical direction. These forests developed in an area where the virgin forests suffered clear-cutting. After excluding habitat heterogeneity, we divided two plots into upper and lower layers according to the tree height, and then analyzed the spatial pattern, species distribution, and size differentiation using the pair correlation function g(r) and the mark correlation function (MCF) kmm (r), respectively. The following key results were obtained: (1) the upstory was slightly clustered, whereas the understory had an intensively clumped pattern. An uneven pattern of germplasm resources in the early stages of succession and seed dispersion limitation may have contributed to the aggregation of tree species. (2) The spatial correlation among the main populations in each layer, and between both layers, had a largely random association, suggesting that differences in tree growth and physiological characteristics play an important role in species association. (3) Species aggregation decreased as the observation scale increased; however, the aggregation intensity of the understory was significantly higher than that of the upstory, which may be related to negative density dependence and niche complementarity. (4) Size differentiation in the upstory was significant, with small trees gathered together. There were no obvious differences in tree size in the lower layer, indicating a higher competitive pressure among trees in the upper layer. In conclusion, the spatial structure of trees in terms of vertical stratification differs, which is of great significance for investigating the mechanisms by which species coexist.