The present work focuses on a local survey of free-living amoebae (FLA) that cause opportunistic and nonopportunistic infections in humans. Determining the prevalence of FLA in water sources can shine a light on the need to prevent FLA related illnesses. A total of 150 samples of tap water were collected from six districts of Sivas province. The samples were filtered and seeded on nonnutrient agar containing Escherichia coli spread. Thirty-three (22%) out of 150 samples were found to be positive for FLA. The FLA were identified by morphology and by PCR using 18S rDNA gene. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of three different species, Acanthamoeba castellanii, Acanthamoeba polyphaga, and Hartmannella vermiformis. Naegleria fowleri, Balamuthia mandrillaris, or Sappinia sp. was not isolated during the study. All A. castellanii and A. polyphaga sequence types were found to be genotype T4 that contains most of the pathogenic Acanthamoeba strains. The results indicated the occurrence and distribution of FLA species in tap water in these localities of Sivas, Turkey. Furthermore, the presence of temperature tolerant Acanthamoeba genotype T4 in tap water in the region must be taken into account for health risks.