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Free-living amoebae (FLA) thrive in diverse environmental conditions. The present study aimed to define the FLA distribution from the Caspian Sea as well as from hospital ward dust from Guilan, Iran. Seawater (20) and hospital ward dust samples (100) were collected from May to June 2018. Seawater samples were vacuum filtered through a 0.45 μm pore-size membrane. Dust was collected using sterile gauze, washed with sterile distilled water, with washings collected thereafter. Washings were similarly filtered as seawater samples. FLA from the filtered material was cultivated in non-nutrient agar. Molecular analysis was performed by PCR and sequencing using specific primers for Acanthamoeba, Naegleria, and Vermamoeba/Hartmanella. Culture and PCR returned 50 and 65% positivity, respectively, for seawater samples where sequencing revealed Acanthamoeba T2, T5 and T6 genotypes and A …