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Abstract Objective These amoebas can cause dangerous illnesses when they accidentally enter the human body, so it is necessary to determine various forms of organisms in water resources to prevent the danger they can cause and risks to human health. Currently, in Bandar Abbas, there is no sufficient information about the distribution of Acanthamoeba, and we intended to study its frequency and determine the related genotypes. Results Out of 83 water samples collected from different resources in the city, 31 plates (37.3%) were found to be positive for free-living amoebae. Of these, five were identified as Acanthamoeba (6%) by culture method and 8 (9.6%) by molecular method. Positive sample sequence analysis enabled us to distinguish two genotypes of T4 (7 cases) and T15 (1 case) in this study.