Acanthamoeba spp. are ubiquitous free-living amoeba with genotypes that cause severe pathology of the eyes, central nervous systems, and rare reports of cutaneous infections. The Seven Crater Lakes are freshwater water resources in Laguna, Philippines primarily used for aquaculture and tourism. A total of 16 surface water samples were collected from different sampling areas per Crater Lake and placed in sterile plastic containers. Samples were filtered using 1.2 μm pore size, glass microfiber filter. Filtered sediments were placed on non-nutrient agar lawned with Escherichia coli and incubated aerobically at 35 °C for 14 days. Six (6) out of 16 water samples exhibited amoebic growth. Cystic stages revealed circular to stellate morphology under light microscopy which were initially classified as Acanthamoeba spp. DNA from positive isolates were made to react with polymerase chain reaction using Acanthamoeba specific primers JDP1 5′-GGCCCAGATCGTTTACCGTGAA-3′ and JDP2 5′-TCTCACAAGCTGCTAGGGAGTCA-3′conﬁrmed the presence of several Acanthamoeba species. Phylogenetic analysis revealed the presence of seven isolates belonging to Acanthamoeba genotypes T4, T5, and T9. The presence of potentially pathogenic Acanthamoeba genotypes in The Seven Crater Lakes of Laguna signifies risk to human health which necessitates the development of programs, policies, and guidelines on the understanding, prevention, and management of potential human infections.