<p>Free living amoebas are widespread protozoans that can be found in different environmental resources such as water bodies, soil and clays. Free living amoebae particularly <em>Acanthamoeba</em> could lead to diseases with poor prognosis including ameobic keratitis (AK) and granulomatous amebic encephalitis (GAE) especially in contact lens wears and immunocompromised individuals, respectively. In the present study 50 samples were collected from water resources (hot-springs, cold springs, rivers, wells, refined drinking water and subterranean canals) of Kaleybar and Khodaafarin, East Azarbayjan Province, Iran. The samples were transferred to laboratory and cultured on 1.5% non-nutrient agar medium containing <em>Escherichia coli</em> bacteria at room temperature and finally examined by microscopic methods. The results showed that out of 50 water samples, 40% were positive samples for free-living amoebae (FLA) including 60% <em>Acanthamoebae</em> genus, %35 <em>Haetmannellidae</em>, %20 <em>Valkamphiids</em>, %20 <em>Vannella</em>, %10 <em>Thechamoeba</em> (45% of plates had mix contamination). The present research was the first study to address the presence of potentially pathogenic free living amoebae in Kaleybar and Khodaafarin, East-Azerbaijan Province.</p>