Despite much interest in regulatory evolution, how promoters have evolved remains poorly studied, mainly owing to paucity of data on promoter regions. Using a new set of high-quality experimentally determined core promoters of the human genome, we conducted a comparative analysis of 2,492 human and rhesus macaque promoters and their neighboring nearly neutral regions. We found that the core promoters have an average rate of nucleotide substitution substantially higher than that at 4-fold degenerate sites and only slightly lower than that for the assumed neutral controls of neighboring noncoding regions, suggesting that core promoters are subject to very weak selective constraints. Interestingly, we identified 24 core promoters (at false discovery rate = 50%) that have evolved at an accelerated rate compared with the neutral controls, suggesting that they may have undergone positive selection. The inferred positively selected genes show strong bias in molecular function. We also used population genetic approaches to examine the evolution of core promoters in human populations and found evidence of positive selection at some loci. Taken together, our results suggest that positive selection has played a substantial role in the evolution of transcriptional regulation in primates.