This study aimed to detect the presence of Acanthamoeba spp. in different water resources of Zahedan, southeast of Iran, and also systematically reviewed all publications regarding Acanthamoeba in Iran (2005–2018). Fifty water samples were collected from different water resources in Zahedan. The positive samples were identified morphologically and subjected to PCR using fragments of 18S rRNA. In the systematic review, data collection using particular terms was carried out using the following electronic databases including Science Direct, ISI Web of Science, MEDLINE, EBSCO, Scopus, and Google Scholar. A total of 17 (34%) samples were positive for Acanthamoeba spp., and nucleotide sequencing indicated that 15 samples (88.23%) belonged to the T4 genotype and the rest belonged to the T5 genotype. A total of 39 studies reported genotyping of Acanthamoeba spp. from various geographical areas of Iran and revealed that T4 (35 studies), T5 (19 studies), T3 (11 studies), T11 (8 studies), and T2 (6 studies) genotypes were the most prevalent in Iran. The T4 genotype of Acanthamoeba is a prevalent free-living amoeba and widely distributed not only in Zahedan but also in other provinces of Iran. Phylogenetic analysis reveals that A. castellanii and A. griffini predominantly colocalize with the T4 genotype.