The two-domain system is a biological classification of all organisms in the tree of life into two domains: Archaea, which includes eukaryotes in this classification, and Bacteria. It emerged from development of knowledge of archaea diversity and challenges the widely accepted three-domain system that classifies life into Bacteria, Archaea, and Eukarya. It was preceded by the eocyte hypothesis of James A. Lake in the 1980s, which was largely superseded by the three-domain system, due to evidence at the time. Better understanding of archaea, especially of their roles in the origin of eukaryotes through symbiogenesis with bacteria, led to the revival of the eocyte hypothesis in the 2000s. The two-domain system became more widely accepted after the discovery of a large kingdom of archaea called Promethearchaeati in 2017, which evidence suggests to be the evolutionary root of eukaryotes, thereby making eukaryotes members of the domain Archaea.

While the features of promethearchaea do not completely rule out the three-domain system, the notion that eukaryotes originated within Archaea has been strengthened by genetic and proteomic studies. Under the three-domain system, Eukarya is mainly distinguished by the presence of "eukaryotic signature proteins" that are not found in Archaea and Bacteria. However, promethearchaea contain genes that code for multiple such proteins.

Classification of life into two main divisions is not a new concept, with the first such proposal by French biologist Édouard Chatton in 1938. Chatton distinguished organisms into:

While he coined the terms in 1925, his detailed classification was published in a limited circulation format in 1938, titled Titres et Travaux Scientifiques (1906–1937) de Edouard Chatton. These were later named empires, and Chatton's classification as the two-empire system. Chatton used the name Eucaryotes only for protozoans, excluded other eukaryotes, and published in limited circulation so that his work was not recognised. His classification was rediscovered by Canadian bacteriologist Roger Yates Stanier of the University of California in Berkeley in 1961 while at the Pasteur Institute in Paris. The next year, Stanier and his colleague Cornelis Bernardus van Niel published in Archiv für Mikrobiologie (now Archives of Microbiology) Chatton's classification with Eucaryotes eloborated to include higher algae, protozoans, fungi, plants, and animals. It became a popular system of classification, as John O. Corliss wrote in 1986: "[The] Chatton-Stanier concept of a kingdom (better, superkingdom) Prokaryota for bacteria (in the broadest sense) and a second superkingdom Eukaryota for all other organisms has been widely accepted with enthusiasm."

In 1977, Carl Woese and George E. Fox classified prokaryotes into two groups (kingdoms), Archaebacteria (for methanogens, the first known archaea) and Eubacteria, based on their 16S ribosomal RNA (16S rRNA) genes. In 1984, James A. Lake, Michael W. Clark, Eric Henderson, and Melanie Oakes of the University of California, Los Angeles described what was known as "a group of sulfur-dependent bacteria" as a new group of organisms called eocytes (for "dawn cells") and created a new kingdom Eocyta. With it they proposed the existence of four kingdoms, based on the structure and composition of the ribosomal subunits, namely Archaebacteria, Eubacteria, Eukaryote and Eocyta Lake further analysed the rRNA sequences of the four groups and suggested that eukaryotes originated from eocytes, and not archaebacteria, as was generally assumed. This was the basis of the eocyte hypothesis. In 1988, he proposed the division of all life forms into two taxonomic groups:

In 1990, Woese, Otto Kandler, and Mark Wheelis showed that archaea are a distinct group of organisms and that eocytes (renamed Crenarchaeota as a phylum of Archaea but corrected as Thermoproteota in 2021) are Archaea. They introduced the major division of life into the three-domain system comprising domain Eucarya, domain Bacteria, and domain Archaea. With a number of revisions of details and discoveries of several new archaea lineages, Woese's classification gradually gained acceptance as "arguably the best-developed and most widely-accepted scientific hypotheses [with the five-kingdom classification] regarding the evolutionary history of life."

The three-domain concept did not, however, resolve the issues with the relationship between Archaea and eukaryotes. As Ford Doolittle, then at the Dalhousie University, put it in 2020: "[The] three-domain tree wrongly represents evolutionary relationships, presenting a misleading view about how eukaryotes evolved from prokaryotes. The three-domain tree does recognize a specific archaeal–eukaryotic affinity, but it would have the latter arising independently, not from within, the former."

The two-domain system relies mainly on two key concepts that define eukaryotes as members of the domain Archaea and not as a separate domain: eukaryotes originated within Archaea, and promethearchaea represent the origin of eukaryotes.