Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes. The present study combines the description of strains isolated from fishes with presentation of an improved method for subgeneric classification. Acanthamoeba spp. were isolated aseptically from tissues of 14 (1.7%) of 833 asymptomatic fishes collected in rivers and streams in the Czech Republic. Acanthamoebae successfully cloned from 10 of the 14 isolated strains were examined here. Morphology of these isolates was evaluated using light optics plus scanning and transmission electron microscopy. Cyst morphology, which varied extensively within and among clones, was most like morphological group II, but species-level classification was considered impossible. A distance analysis based on 442 bases in an 18S rDNA polymerase chain reaction fragment of about 460 bp placed the isolates in a clade composed of sequence types T3, T4, and T11, the 3 subdivisions of morphological group II. Fluorescent in situ hybridization (FISH) using oligonucleotide probes indicated that all isolates belong to a single subdivision of group II, the T4 sequence type. It has been concluded that the fish isolates are most closely related to strains commonly isolated from human infections, especially Acanthamoeba keratitis. The shorter diagnostic fragment sequences have proved nearly as useful as complete 18S rDNA sequences for identification of Acanthamoeba isolates.