Free-living amoebae (FLA) are found widely in soil and water in the nature. Among them in which potentially pathogenic for humans and animals are known as "potential pathogenic free-living amoebae (PPFLA)". PPFLA are characterized as the causes of clinical manifestations leading to death especially in immunosuppressed people. Four genus of PPFLA (Acanthamoeba, Naegleria, Balamuthia and Sappinia) are known to be pathogenic to humans. The aims of this study were to investigate the presence of PPFLA in the water supplies in Turkey and to determine their in vivo pathogenicity. A total of 664 water samples were collected from the ponds, rivers, streams and wells found in provinces located at different regions (central, western, eastern and southeastern regions) of Turkey. These samples were initially inoculated in the monoxenic culture media and evaluated by both microscopy and polymerase chain reaction (PCR) in terms of the presence of FLA. The samples identified as positive were then cultured in axenic media, the growth of amoebae that were confirmed microscopically, were than studied with PCR for molecular characterization. The isolates that were found positive by PCR from axenic cultures were inoculated intranasally to immunocompetent and immunodeficient (athymic) [BALB/c Rag2(-/-) gamma(c)(-/-)] BALB/c mice followed by the evaluation on the 21st day by histopathological and molecular methods to investigate their in vivo pathogenicity. In our study, 143 water samples were detected as positive in monoxenic cultures and 41 of them were detected as positive in axenic cultures. Twenty of 41 samples detected as positive in axenic culture could be continued in culture for three months. As a result of PCR using primers common to SYA, only nine have been identified from 20 samples as positive. According to the result of the PCR with specific primers, all (n=9) were positive for Acanthamoeba sp., eight for Sappini sp. and five for Balamuthia mandrillaris, while none was observed Naegleria fowleri. Histopathologic examination revealed that both groups of mice that were infected with the nine isolates had normal brain tissue sections; but haemorrhages and mononuclear cell proliferation were determined in four immunocompetent and seven athymic animal lung sections. When the presence of parasites in tissue samples were evaluated by real-time PCR, Balamuthia was detected in at least one blood, lung, brain or nasal mucosa sample of the four immunocompetent mice, Sappinia sp. in four and Acanthamoeba sp. in seven immunocompetent mice infected with nine isolates. Additionally, seven Balamuthia sp., seven Sappinia sp. and eight Acanthamoeba sp. were detected in immunodeficient mice. In this study, B. mandrillaris and Sappinia sp. were the first isolated potentially pathogenic amoebae from water supplies located at different parts of Turkey. As a result awareness and precautions against suspicious water supplies used for drinking, daily use and swimming purposes should be treated more carefully.