Acanthamoeba spp. are free-living amoeba commonly found in environmental sources such as soil, water, and dust. This ubiquitous amoeba is the causative agent of amoebic keratitis (AK) and encephalitis. The present study aimed to investigate the presence of Acanthamoeba spp. in the water sources of Kermanshah city, Iran. Sixty water samples were taken from different localities of Kermanshah including agricultural canals, rivers, and swimming pools. Filtration and cultivation were carried out on non-nutrient agar medium (NNA). The axenic cultivation was performed for all of the positive isolates. PCR analysis was performed on positive samples. Sequencing was done for 12 PCR products. Genotypes were identified by blast search and homology analysis. The obtained data were analyzed using Statistical Package for the Social Sciences (SPSS 16) software. Acanthamoeba spp. was found in 46 (76.66%) water samples and amoebae were grown in the TYI-S-33 medium. Sequencing of 12 samples proved that Acanthamoeba belonged to T4 (75%), T2 (8.34%), T5 (8.33%) and T11 (8.33%) genotypes. In this study, Acanthamoeba T4 (75%), T2 (8.34%), T5 (8.33%) and T11 (8.33%) genotypes were isolated from the water of Kermanshah city. Thus, hygiene consideration is recommended to prevent the contamination.