Abstract: Evolution of new cellular functions can be achieved both by changes in protein coding sequences and by alteration of expression patterns. Variation of expression may lead to changes in cellular function with relatively little change in genomic sequence. We therefore hypothesize that one of the first signals of functional divergence should be evolution of transcription factor-binding sites (TFBSs). This adaptation should be detectable as substantial variation in the TFBSs of alleles. New data sets allow the first analyses of intraspecies variation from large number of whole-genome sequences. Using data from the Saccharomyces Genome Resequencing Project, we have analyzed variation in TFBSs. We find a large degree of variation both between these closely related strains and between pairs of duplicated genes. There is a correlation between changes in promoter regions and changes in coding sequences, indicating a coupling of changes in expression and function. We show that 1) the types genes with diverged promoters vary between strains from different environments and 2) that patterns of divergence in promoters consistent with positive selection are detectable in alleles between strains and on duplicate promoters. This variation is likely to reflect adaptation to each strain's natural environment. We conclude that, even within a species, we detect signs of selection acting on promoter regions that may act to alter expression patterns. These changes may indicate functional innovation in multiple genes and across the whole genome. Change in function could represent adaptation to the environment and be a precursor to speciation.