Acanthamoeba as free-living parasites are scattered ubiquitously, throughout the world. This study was aimed to evaluate the presence of Acanthamoeba spp. genotypes in the recreational water sources in Gorgan County, the capital of Golestan Province using both morphological and molecular approaches. Thirty water samples were collected from different recreational waters in Gorgan, the capital of Golestan Province, northern Iran during 2015-2016. Samples were filtered and followed by culture in non-nutrient agar. Acanthamoeba were identified both by morphological and molecular analysis. The pathogenical potential of positive cloned samples were also determined using tolerance test. Twenty-six percent of recreational water were identified as Acanthamoeba spp. based on the morphological analysis and from these positive samples, five samples were successfully sequenced after molecular studies. Phylogenetic analysis showed the clustering of four samples in T4 genotype group and only one sample as T15 genotype. Thermotolerance test revealed that all cloned samples were highly positive. Since the attractiveness of recreational places for people is increasing, the potential risk of this water should be monitored routinely in each region. More studies are needed to better evaluate the risk of this ubiquitous parasite for the human.