Yellowfin goby Acanthogobius flavimanus affected with X-cell pseudotumors were sampled from a river estuary in Tokyo Bay, Japan. We amplified the gene for small subunit ribosomal RNA (18S rRNA) of X-cells of the goby with PCR using universal primers. The gene that we obtained (DDBJ Accession no. AB451874) showed 91% sequence identity to that of the X-cells of the flathead flounder Hippoglossoides dubius. With in situ hybridization, the probes specific for the gene that we obtained hybridized with the goby X-cells but not with the flounder X-cells, whereas probes for the 18S rRNA gene of flounder X-cells hybridized with the flounder X-cells but not with goby X-cells. These findings indicate that, although the X-cells found in the goby are closely related to the protist found in flounder, the two are clearly distinct organisms.