We have applied a comparative phylogenomic analysis to study the evolutionary relationships between GC content, CpG-dinucleotide content (CpGs), potential nuclear factor I (NFI) binding sites, and potential Z-DNA forming regions (ZDRs) as representative structural and functional GC-rich genomic elements. Our analysis indicates that CpG and NFI sites emerged with a general accretion of GC-rich sequences downstream of the eukaryotic transcription start site (TSS). Two distinct classes of ZDRs are observed at different locations proximal to the eukaryotic TSS. A robust CA/TG class of ZDRs was seen to emerge upstream of the TSS and independently of GC content, CpGs, and NFIs, whereas a second, weaker CG type appears to have evolved along with these downstream GC-rich elements. Taken together, the results provide a model for how GC-rich structural and functional eukaryotic markers emerge relative to each other, and indicate two distinct transition points for their occurrence: the first at the pro/eukaryotic boundary, and the second at or near the amniotic boundary.evolution ͉ genomic analysis ͉ Z-DNA