DNA replication origins are poorly characterized genomic regions that are essential to recruit and position the initiation complex to start DNA synthesis. Despite the lack of specific replicator sequences, initiation of replication does not occur at random sites in the mammalian genome. This has lead to the view that DNA accessibility could be a major determinant of mammalian origins. Here, we performed a high-resolution analysis of nucleosome architecture and initiation sites along several origins of different genomic location and firing efficiencies. We found that mammalian origins are highly variable in nucleosome conformation and initiation patterns. Strikingly, initiation sites at efficient CpG island-associated origins always occur at positions of high-nucleosome occupancy. Origin recognition complex (ORC) binding sites, however, occur at adjacent but distinct positions marked by labile nucleosomes. We also found that initiation profiles mirror nucleosome architecture, both at endogenous origins and at a transgene in a heterologous system. Our studies provide a unique insight into the relationship between chromatin structure and initiation sites in the mammalian genome that has direct implications for how the replication programme can be accommodated to diverse epigenetic scenarios.