A comprehensive survey was conducted along 10 km of geothermal rivers in southwestern Iran. A total of 40 water samples were tested for the presence of Acanthamoeba spp., and genotypes were determined by targeting the diagnostic fragment 3 region of the 18S rRNA gene. The pathogenic potential of all positive isolates was also identified using tolerance ability test. High occurrences of Acanthamoeba (50%) were detected in the sampling areas. Based on sequencing analysis, isolates belonging to T4 (93.7%) and T2 (6.25%) genotypes were reported. Thermo- and osmotolerance tests revealed that five strains are highly pathogenic. Since every collection site of this study was associated with high human activity, posting of warning signs, monitoring of recreational water sources, and awareness of high-risk people are of utmost importance. To the best of our knowledge, the present research is the first to report T2 genotype from geothermal water sources in Iran.