Regulation of gene expression is a complex affair involving changes in chromatin structure and histone modifications, as well as coordinated action amongst trans-acting transcription factors (TF). To date the extent to which variation in these regulatory mechanisms can influence gene expression, and thus contribute to phenotypic variation, has been limited to examining each regulatory layer in isolation or in the case of TFs, just one or two factors. Here, we present a unified analysis of the regulatory network in five diverse strains of the yeast S. cerevisiae, including genome-wide measurements of the binding locations of 52 TFs (~25% of all TFs), chromatin accessibility and four histone modifications. We observed a broad-range (between 0.1 and 0.75) in the fraction of binding sites different between strains across all TFs, with highly conserved and essential TFs displaying significantly lower binding variability. Variable binding regions and chromatin states were enriched for single-nucleotide variants, and coordinated changes across multiple regulatory layers, in contrast to any single data type, were highly correlated with differences in gene expression. Our data provide a unique resource to examine the impact of sequence-level variation across multiple layers of gene regulation.