Background and Aim: Acanthamoeba is a free-living amoebae commonly found in the environmental sources such as soil, water, and dust. This parasite is the causative agent of amoebic keratitis (AK). The objective of the present study was to investigate the presence of Acanthamoeba genotypes in water sources in Bojnurd City. Materials and Methods: Totally, 50 samples of water were taken from different localities of Bojnurd including agricultural canals, rivers, and swimming pools. Filtration and cultivation were carried out on non-nutrient agar medium (NNA). PCR analysis was conducted on positive samples. Sequencing was done for 10 PCR products. Genotypes were identified by means of Blast search and homology analysis. The obtained data was analyzed using SPSS software (V:16). Results: Acanthamoeba amoebae was found in 34 (68%) samples of the water. Genotyping of 10 samples proved to be T4(100%) genotype. Conclusion: While Acanthamoeba amoebae is found in our surrounding environment which unknowingly contaminates us every day. Thus, to prevent the contamination, hygiene consideration is recommended.