Free-living Amoebae of Acanthamoeba genus include non-pathogenic and pathogenic strains that are currently classified in 18 different genotypes, T1-T18. In this study, a survey was carried out to evaluate the presence of Acanthamoeba strains in soil samples collected between 2012 and 2013 in Gran Canaria Island, Canary Islands, Spain. Samples were inoculated onto non-nutrient agar (NNA) plates and were checked for the presence of Acanthamoeba. Identification of Acanthamoeba strains was based on the morphology of the cyst and trophozoite forms. Subsequently, positive samples were cloned for their molecular characterization at the genotype level by sequencing the DF3 region located in the 18S rDNA gene of Acanthamoeba as previously described. Sequencing results revealed the presence of T2, T5 and T4 genotypes within the studied samples. To the best of our knowledge, this is the first report demonstrating the presence of Acanthamoeba in Gran Canaria Island and the first study at the genotype level in the Canary Islands.