OBJECTIVE A previous study confirmed the contamination of water sources with this parasite in Arak, Markazi Province, Iran. The current study investigated soil sources and determined the predominant genotype of Acanthamoeba in this region of Iran. METHODS Forty-eight soil samples, collected from different regions of Arak, Markazi province, Iran, were evaluated in this study. The samples were processed and identified by culturing on a specific medium, performing PCR assay, and sequencing the PCR products. Finally, using the NCBI database, the genotypes were determined. RESULTS Of 48 soil samples, 33.3% and 31.25% were contaminated with Acanthamoeba according to the culture and molecular assays, respectively. The majority of these isolates belonged to the T4, T5 and T6 genotypes of Acanthamoeba. CONCLUSIONS The genotypes of most isolates from soil samples in Arak similar to other regions of Iran belong to T4 genotype of this parasite. New sequence accession numbers include MG066681 and MG298785-MG298794.