Current diagnostic methods for Acanthamoeba identification rely heavily on light microscopic techniques that do not provide sufficient information about the identification of Acanthamoeba at the species level, thus delaying accurate identification of the infective agent. Here we report the use of polymerase chain reaction (PCR)-based restriction enzyme analyses to detect and speciate Acanthamoeba from both clinical and environmental sources by comparing their restriction endonuclease patterns. Significant diversity was observed between and within morphologically defined Acanthamoeba species. The usefulness of PCR-based assays and other available diagnostic methods is discussed.