the packaging of eukaryotic genomes into nuclesomes plays critical roles in chromatin organization and gene regulation studies in saccharomyces cerevisiae indicate that nucleosome occupancy is partially encoded by intrinsic antinucleosomal dna sequences such as poly a sequences as well as by binding sites for trans acting factors that can evict nucleosomes such as reb1 and the rsc3 30 complex here we use genome wide nucleosome occupancy maps in 13 ascomycota fungi to discover large scale evolutionary reprogramming of both intrinsic and trans determinants of chromatin structure we find that poly g s act as intrinsic antinucleosomal sequences comparable to the known function of poly a s but that the abundance of poly g s has diverged greatly between species obscuring their antinucleosomal effect in low poly g species such as s cerevisiae we also develop a computational method that uses nucleosome occupancy maps for discovering trans acting general regulatory factor grf binding sites our approach reveals that the specific sequences bound by grfs have diverged substantially across evolution corresponding to a number of major evolutionary transitions in the repertoire of grfs we experimentally validate a proposed evolutionary transition from cbf1 as a major grf in pre whole genome duplication wgd yeasts to reb1 in post wgd yeasts we further show that the mating type switch activating protein sap1 is a grf in s pombe demonstrating the general applicability of our approach our results reveal that the underlying mechanisms that determine in vivo chromatin organization have diverged and that comparative genomics can help discover new determinants of chromatin organization.