<p>The last eukaryotic common ancestor primarily inherited its core genetic system from archaea. However, it remains unclear when and how these essential machineries expanded their compositional and regulatory sophistication during eukaryogenesis. Here we combine statistical, phylogenetic, structural and biochemical approaches to examine the compositional diversity of the DNA replication machinery, that is, the replisome, across archaea and eukaryotes. We find that different lineages of Asgard archaea encode distinct replisome components with eukaryotic signatures, including a Baldrarchaeia-encoded DNA polymerase δ-like complex, a Sif/Wukong/Heimdallarchaeia-encoded primase complex and a Lokiarchaeales-encoded RFC clamp-loader complex. Copy number expansions driven by horizontal gene transfer probably contributed to the structural diversification of Asgard archaeal replisomes, including phylogenomic markers RfcS and Fen1, which were previously presumed to be transmitted vertically. Our analyses suggest that these distributed innovations were sequentially acquired by the early eukaryotic ancestor before the burst of gene duplications leading to the last eukaryotic common ancestor. By placing the captured events of gene gain and loss within the context of archaea–eukaryote evolution—as inferred from the phylogeny of concatenated single-copy replisome genes—we propose a hypothetical model for the emergence of the complex eukaryotic replisome.</p>