Significance Nucleosomal modifications have been implicated in fundamental epigenetic regulation, whereas the roles of nucleosome binding in shaping changes through evolution remain to be addressed. Here we performed a comparative study to clarify the roles of nucleosome occupancy in exon origination. By profiling a high-resolution, cross-species mononucleosome landscape for mammalian tissues, we found nucleosome occupancy profiles are conserved across tissues and species. Further, through a phylogenetic approach, we found that the feature of differential nucleosome occupancy appears prior to the origination of new exons and, presumably, facilitates the origin of new exons by increasing the splice strength of the ancestral nonexonic regions through driving a local difference in GC content, which suggests the function of nucleosome binding in exonization.