Members of the Marseilleviridae family are large DNA viruses with icosahedral particle structures that infect Acanthamoeba cells. The first Marseillevirus to be discovered was isolated in 2009. Since then, several other members of the Marseilleviridae family have been reported, including Lausannevirus, Senegalvirus, Cannes 8 virus, Insectomime virus, Tunisvirus, Melbournevirus, Port-Miou virus, and Brazilian Marseillevirus, which have been isolated from Europe, Africa, Australia, and South America. The morphological and genomic properties of a new Marseilleviridae family member, Tokyovirus, discovered in a water/soil sample from a Japanese river in Tokyo, were described in the present study. Tokyovirus possesses icosahedral particles of up to 200 nm in diameter, as revealed by a transmission electron microscopy (TEM) analysis, which form a giant virion factory in Acanthamoeba cells. A preliminary genome analysis predicted 487 coding sequences. A dot plot analysis and phylogenetic analysis using family B DNA polymerase, proliferating cell nuclear antigen (PCNA), and DNA-directed RNA polymerase alpha subunit genes revealed that Tokyovirus shares similarities with Marseillevirus, Melbournevirus, and Cannes 8 virus (Marseilleviridae subclade A), but not with Lausannevirus and Port-Miou virus (subclade B), Tunisvirus and Insectomime virus (subclade C), or Brazilian Marseillevirus (subclade D), suggesting that Tokyovirus has evolved separately from the previously described Marseilleviridae members.