The members of Acanthamoeba genus are ubiquitous amoeba which could be a pathogenic parasite. The amoeba is resistant to the common chlorine concentration that used for disinfecting the swimming pool water. Therefore, the pools can be suitable environments for the survival and multiplication of the amoeba. In this cross sectional study, 10 indoor recreational water centers from different regions of Tabriz city were selected and sampling was done from fixed and floating biofilms of the swimming pools and hot tubs. The samples were cultured and monitored for the presence of amoeba cyst or trophozoite. For molecular identification of Acanthamoeba, PCR (polymerase chain reaction) and sequencing were conducted based on genus specific fragment of 18S ribosomal DNA (Rns). Acanthamoeba contamination was observed in 6 centers of 10 recreational centers. Based on the amoeba isolation from fixed and floating biofilms, 2 (20%) swimming pools, and 5 (50%) hot tubs were contaminated. Based on the type of the sample, the highest contamination was found in the hot tub water (40%) and the least was found in the swimming pools water (10%) and fixed biofilms of the swimming pools (10%). Out of 8 isolates, 5 (62.5%) were shown expected product in PCR amplification. Sequence analysis showed that Acanthamoeba isolates belonged to the T3 and T4 genotypes. The study revealed a high degree of contamination in the indoor recreational water centers in Tabriz city. So, it is essential to pay closer attention to the hygiene of swimming pools and hot tubs.