Background: Different genotypes of Acanthamoeba have been abundantly isolated in environmental samples such as water, soil, and dust, as well as in different hospital departments and eyewash stations. This protozoan is a potential hazard for immunocompromised patients and contact lens wearers. The aim of the present study was isolation and genotyping of environmental and corneal isolates of Acanthamoeba in Hamadan, west of Iran. Methods: During 2018-2020, a total of 104 environmental samples including, water, soil, and dust and 16 corneal SCRAPING samples were collected and investigated for the presence of Acanthamoeba using morphological and molecular identification tools. Genotypes were determined using sequence analysis of the diagnostic fragment 3 (DF3) from Acanthamoeba-specific amplimer S1 (ASA. S1) gene. Phylogenetic tree was constructed with the MEGA7 software using Neighbor-Joining method. Results: The presence of Acanthamoeba spp. was determined in 87. 5% of water, 53. 1% of soil, and 25% of dust samples. From 30 dust samples collected from eight wards of three hospitals, 7 (23. 3%) were contaminated with Acanthamoeba. Sequencing analysis of environmental samples revealed that the T4 genotype was the most prevalent (92. 6%) one. Genotypes T2 (1. 9%), T2/T6 (1. 9%), and mixed T4 and T2/T6 (3. 7%) were also identified in environmental samples. Acanthamoeba was seen in none of the examined corneal SCRAPING samples from patients with suspected keratitis. Conclusion: The widespread occurrence of this potentially pathogenic amoeba in most hospital wards and environmental resources and areas of the region highlights a strong need to increase awareness regarding this ubiquitous amoeba among susceptible individuals, such as immunocompromised patients and contact lens wearers.