Acanthamoeba is a free-living protozoan genus found in a wide variety of natural habitats, including water, soil, and air. Pathogenic isolates of Acanthamoeba are medically relevant as the causative agent of sight-threatening Acanthamoeba keratitis (AK), serious infections of other organs, and fatal granulomatous amebic encephalitis. A total of 197 sets of contact lens care system and bathroom basin samples from contact lense users were cultured for the isolation of Acanthamoeba. In addition, Acanthamoeba isolates obtained, between September 2006 till June 2008, from cornea scraping of patients suspected of amebic kerattitis were also included in this study. Genotype of the isolates were identified by 18S rRNA gene sequence analysis. We obtained 17 Acanthamoeba isolates from bathroom basin of contact lens users and 7 isolates from patients with keratitis in National Taiwan University Hospital. Acanthamoeba was not detected in contact lens box, normal saline, contact lens care fluid and disposable contact lenses. Among the 24 isolates, 19 were identified as Rns genotype T4 , 3 were Rns genotype T3 and 2 Rns genotype T6. Thus most Acanthamoeba strain isolated from home water supplies of contact lens users and patients with keratitis in National Taiwan University Hospital belong to Rns genotype T4, which agree with findings of similar studies abroad.