Despite its potential role in the evolution of complex phenotypes, the detection of negative (purifying) and positive selection on noncoding regulatory sequence has been elusive because of the inherent difficulty in predicting the functional consequences of mutations on noncoding sequence. Because the functioning of regulatory sequence depends upon both chromatin configuration and cis-regulatory factor binding, we investigate the idea that the functional conservation of regulatory regions should be associated with the conservation of sequence-dependent bending properties of DNA that determine its affinity for the nucleosome. Recent advances in the computational prediction of sequence-dependent affinity to nucleosomes provide an opportunity to distinguish between neutral and nonneutral evolution of fine-scale chromatin organization. Here, a statistical test is presented for detecting evolutionary conservation and/or adaptive evolution of nucleosome affinity from interspecies comparisons of DNA sequences. Local nucleosome affinities of homologous sequences were calculated using 2 recently published methods. A randomization test was applied to sites of mutation to evaluate the similarity of DNA-nucleosome affinity between several closely related species of Saccharomyces yeast. For most of the genes we analyzed, the conservation of local nucleosome affinity was detected at a few distinct locations in the upstream noncoding region. Our results also demonstrate that different patterns of chromatin evolution have shaped DNA-nucleosome interaction at the core promoters of TATA-containing and TATA-less genes and that elevated purifying selection has maintained low affinity for nucleosome in the core promoters of the latter group. Across the entire yeast genome, DNA-nucleosome interaction was also discovered to be significantly more conserved in TATA-less genes compared with TATA-containing genes.