In this study, a quantitative real-time PCR was developed to detect and quantify Acanthamoeba spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and three thermal spring recreation areas. The overall detection rate was 14.2 % (25/176) for Acanthamoeba spp. The percentages of samples containing Acanthamoeba spp. from river water, raw drinking water, and thermal spring water were 13 % (13/100), 25 % (7/28), and 10.4 % (5/48), respectively. Acanthamoeba spp. concentrations were determined according to SYBR Green quantitative real-time PCR. A plasmid-based standard curve was constructed to determine the Acanthamoeba concentration using dilution factors for achieving 1.36 × 10(9) gene copies per PCR for 18S rRNA gene in Acanthamoeba spp. The resulting concentrations varied by the type of water, in the range of 46-2.6 × 10(2) cells/l in positive raw drinking water, 2.7 × 10(2)-1.5 × 10(4) cells/l in river water, and 54-1.7 × 10(3) cells/l in thermal spring water. The presence of Acanthamoeba spp. in the raw drinking water samples was also found to have a significant difference with heterotrophic plate count. The presence of Acanthamoeba spp. in various aquatic environments may be a potential health hazard and must be further evaluated.