BACKGROUND Free-living amoebae (FLA) are a group of ubiquitous protozoan, which are distributed in the natural and artificial environment sources. The main aim of the current study was to identify the presence of FLA in the recreational hot springs of Sarein in Ardebil Province of Iran. METHODS Seven recreational hot springs were selected in Sarein City and 28 water samples (four from each hot spring) were collected using 500 ml sterile plastic bottles during three month. Filtration of water samples was performed, and culture was done in non-nutrient agar medium enriched with Escherichia coli. Identification of the FLA was based on morphological criteria of cysts and trophozoites. Genotype identification of Acanthamoeba positive samples were also performed using sequencing based method. RESULTS Overall, 12 out of 28 (42.9%) samples were positive for FLA which Acanthamoeba and Vahlkampfiid amoebae were found in one (3.6%) and 11 (39.3%) samples, respectively. Sequence analysis of the single isolate of Acanthamoeba revealed potentially pathogenic T(4) genotype corresponding to A. castellanii. CONCLUSIONS Contamination of hot springs to FLA, such as Acanthamoeba T(4) genotype (A. castellanii) and Vahlkampfiid amoebae, could present a sanitary risk for high risk people, and health authorities must be aware of FLA presence.