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Background: Free-living amoeba (FLA) are wide-spread protozoa that are found in different environmental sources including water, soil, dust, hospital units and ventilation areas. These amoebas can act as opportunistic or nonopportunistic pathogens. Among FLAs, some genera such as Acanthamoeba are important because of their potential pathogenic ability in humans. The purpose of this study is to identify of Acanthamoeba isolated from pond water of parks in a tropical and subtropical region in the Middle East, and its relation with physicochemical parameters.From August to December 2015, 90 samples were collected from pond water of parks of 13 regions of Mashhad City. Physicochemical parameters were measured in situ. After filtering, the samples were cultured on Bacto-agar enriched with Escherichia coli. PCR analysis was conducted on the culture-positive samples, and then the PCR products were sequenced. Statistical analysis was performed by SPSS software and Fisher's Exact and Mann- Whitney test. Results: Among the samples that were examined, 19 samples (21.1%) were positive for Acanthamoeba. The sequencing revealed that Acanthamoeba isolates belonged to T4 genotype of Acanthamoeba. There was no significant relationship between physicochemical parameters and the presence of Acanthamoeba. Conclusion: The prevalence of Acanthamoeba in pond water of parks was relatively high, but there was no significant relationship between physicochemical parameters and the presence of Acanthamoeba. [ABSTRACT FROM AUTHOR]