Purpose The main goal of this study was genotyping of free-living parasites and sub-grouping of pathogenic or non-pathogenic amebae obtained from Turkey's thermal springs. In so doing, distribution and abundance of possible pathogenic or causative strain for humans, which are caused by Acanthamoeba and Naegleria strains, would be elaborated. The number of extensive studies on the general occurrence and distribution of parasitic strains is very high worldwide, but there has been a paucity of information with regard to Turkey. Methods From a total of 434 obtained thermal pool samples, free-living amebas were isolated from 148 water samples using the non-nutrient agar (NNA) culture method. Subsequently, the cultivated samples were used for DNA isolation; then 102 obtained DNA samples were subjected to PCR amplification using various primers for samples of genera Acanthamoeba and Naegleria. Ultimately, estimation of genotype or subtype was evaluated by sequencing. Results About 29 samples that belong to Acanthamoeba and Naegleria were estimated from a total of 102 amplified PCR samples. These eukaryotic PCR products which have Acanthamoeba genus appearance, generated 26 subtypes and 3 Naegleria samples. Among the 26 Acanthamoeba genotypes, 22 aligned sequences were matched with various GenBank reference samples, while the 4 divergent genotypes were not elaborated and marked as ND. Most of the Acanthamoeba genera were determined as likely dominating groups and clustered as T form within totally eight groups. Eight, seven and three subtypes were found as T4A, T15 and T11 genotypes, respectively while the remainings were ultimately found in four groups. Results confirming the predominance of T4A, which is known the most causative form, the presence in the pools. Despite being uncommon, N. fowleri, lovaniensis and australiensis were also observed among the surveyed pools. Conclusion The present study is descriptive and is not unique. However, this is the most comprehensive study of the molecular distribution sampling of thermophilic Acanthamoeba and Naegleria that confirmed and demonstrated their ubiquitous presence throughout Turkey. By this estimation, in some spas, the most and likely causative form Acanthamoeba including T4 and Naegleria fowleri has also been confirmed.