Abstract: Recent studies have characterized significant differences in the cis-regulatory sequences of related organisms, but the impact of these differences on gene expression remains largely unexplored. Here, we show that most previously identified differences in transcription factor (TF)-binding sequences of yeasts and mammals have no detectable effect on gene expression, suggesting that compensatory mechanisms allow promoters to rapidly evolve while maintaining a stabilized expression pattern. To examine the impact of changes in cis-regulatory elements in a more controlled setting, we compared the genes induced during mating of three yeast species. This response is governed by a single TF (STE12), and variations in its predicted binding sites can indeed account for about half of the observed expression differences. The remaining unexplained differences are correlated with the increased divergence of the sequences that flank the binding sites and an apparent modulation of chromatin structure. Our analysis emphasizes the flexibility of promoter structure, and highlights the interplay between specific binding sites and general chromatin structure in the control of gene expression.