Abstract: Gene expression diverges rapidly between related species, playing a key role in the evolution of new phenotypes. The extent of divergence differs greatly between genes and is correlated to promoter nucleosome organization. We hypothesized that this may be partially explained by differential sensitivity of expression to mutations in the promoter region. We measured the sensitivity of 22 yeast promoters with varying nucleosome patterns to random mutations in sequence. Mutation sensitivity differed by up to 10-fold between promoters. This difference could not be explained by the abundance of transcription factor binding sites. Rather, mutation sensitivity positively correlated with the relative occupancy of nucleosomes at the proximal promoter region. Furthermore, mutation sensitivity was reduced upon introduction of a binding site for Reb1, a factor that blocks nucleosome formation, suggesting that nucleosome organization directly regulates mutation sensitivity. Our study suggests an important role for chromatin structure in the evolution of gene expression.