Abstract: DNA replication research to date has focused on model organisms such as the vertebrate Xenopus laevis and the yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. However, animals and fungi both belong to the Opisthokonta, one of about six eukaryotic phylogenetic 'supergroups', and therefore represent only a fraction of eukaryotic diversity. To explore evolutionary diversification of the eukaryotic DNA replication machinery a bioinformatic approach was used to investigate the presence or absence of yeast/animal replisome components in other eukaryotic taxa. A comparative genomic survey was undertaken of 59 DNA replication proteins in a diverse range of 36 eukaryotes from all six supergroups. Twenty-three proteins including Mcm2-7, Cdc45, RPA1, primase, some DNA polymerase subunits, RFC1-5, PCNA and Fen1 are present in all species examined. A further 20 proteins are present in all six eukaryotic supergroups, although not necessarily in every species: with the exception of RNase H2B and the fork protection complex component Timeless/Tof1, all of these are members of anciently derived paralogous families such as ORC, MCM, GINS or RPA. Together these form a set of 43 proteins that must have been present in the last common eukaryotic ancestor (LCEA). This minimal LCEA replisome is significantly more complex than the related replisome in Archaea, indicating evolutionary events including duplications of DNA replication genes in the LCEA lineage which parallel the early evolution of other complex eukaryotic cellular features.