A pathogenic Entamoeba histolytica–like variant (JSK2004 strain) with genetic variations and a novel isoenzyme pattern isolated from a De Brazza's guenon in a Tokyo zoo in Japan has previously been documented. In this study, a multiplex polymerase chain reaction (PCR) assay that could distinguish the JSK2004-type E. histolytica– like variant (JSK04-Eh-V) from E. histolytica and Entamoeba dispar using three newly designed primer sets for amplifying each specific DNA fragment from their small-subunit ribosomal RNA genes was developed and established. Forty-seven primates (11 species) from the zoo were surveyed by multiplex PCR to assess the prevalence of JSK04-Eh-V infection, which was recognized in six individuals of four species, including an Abyssinian colobus monkey, a De Brazza's guenon (including the individual from whom JSK2004 was isolated), a white-faced saki, and a Geoffroy's spider monkey. In addition, the autopsied individuals of an Abyssinian colobus and Geoffroy's spider monkey that died of amoebic liver abscess were also evaluated. DNA samples were also analyzed for specific genotypes based on the nucleotide sequencing of two protein-coding (chitinase and serine-rich E. histolytica protein) genes and the protein-noncoding locus 1-2 that was used for fingerprinting of the E. histolytica strain. These studies indicated that the E. histolytica–like variant infection in this zoo was caused by the same type (i.e., JSK04-Eh-V). An axenic culture medium (yeast extract–iron–maltose–dihydroxyacetone–serum) was developed based on the yeast extract–iron–gluconic acid– dihydroxyacetone–serum medium, which is designed for axenic culture of E. dispar. This new medium could be used for axenically culturing E. histolytica, JSK04-Eh-V, and E. dispar in a single medium.