Background: Acanthamoeba spp. are opportunistic amphizoic protozoans, which are distributed in the natural and artificial environment water sources. This ubiquitous amoeba is the causative agent of Amoebic Keratitis(AK) and Granulomatous Amoebic Encephalitis(GAE). The main aim of the current study was to identify the presence of Acanthamoeba spp. In surface waters of Yasuj district south of Iran. Methods: In this descriptive cross-sectional study, a total of 30 surface water samples were collected from environmental sources, including natural rivers, springs, waterfall and freshwater source in 2013. All samples were collected using 500 ml sterile plastic bottles during two months. After filtration through millipore nylon membrane, samples were cultured on non-nutrient bacto agar medium enriched with Escherichia coli and incubated for 3 to 14 days at room temperature. Identification of the Acanthamoeba spp. was based on morphological criteria of cysts and trophozoites. Following DNA extraction, PCR was used to confirm the microscopically identification. Results: A total of 11 out of 30 samples (36.6%) were positive for Acanthamoeba species based on the morphological criteria. Five out of 11 positive samples (45.46%) were confirmed by PCR method. In total, 5 (16.6%) samples out of 30 samples were positive for Acanthamoeba species based on PCR method. Conclusion: High frequency of Acanthamoeba spp. in different surface water sources specially promenades in that region is an alert for the public health and highlights the needs for more awareness of health professionals and for the related risks.