Evaluating the factors that drive patterns of population differentiation in plants is critical for understanding several biological processes such as local adaptation and incipient speciation. Previous studies have given conflicting results regarding the significance of pollination mode, seed dispersal mode, mating system, growth form, and latitudinal region in shaping patterns of genetic structure, as estimated by FST values, and no study to date has tested their relative importance together across a broad scale. Here we assembled a 337-species dataset for seed plants from publications with data on FST from nuclear markers and species traits, including variables pertaining to the sampling scheme of each study. We used species traits, while accounting for sampling variables, to perform phylogenetic multiple regressions. Results demonstrated that FST values were higher for tropical, mixed-mating, non-woody species pollinated by small insects, indicating greater population differentiation, and lower for temperate, outcrossing trees pollinated by wind. Among the factors we tested, latitudinal region explained the largest portion of variance, followed by pollination mode, mating system and growth form, while seed dispersal mode did not significantly relate to FST. Our analyses provide the most robust and comprehensive evaluation to date of the main ecological factors predicted to drive population differentiation in seed plants, with important implications for understanding the basis of their genetic divergence. Our study is the first that we are aware of to robustly demonstrate greater population differentiation in tropical regions.