Free-living amoebae (FLA) are protists frequently found in water and soils. Some FLA such as Naegleria fowleri, Acanthamoeba and Balamuthia can cause rare but fatal encephalitis. They can also harbor human pathogens (bacteria, viruses and fungi). In 2008, Nf was responsible for the death of a 9-year-old boy who swam in a geothermal bath in Guadeloupe. Our group showed that (i) Nf can be found in most of these hot baths and (ii) soil is the origin of this contamination. Herein, we aimed at analyzing the diversity and distribution of FLA in soil using a targeted metagenomic analysis. A total of 107 soil samples were collected from 27 sites, nearby water ponds, hot springs and rivers. DNA was extracted directly from soil samples or from FLA cultivated at different temperatures (30, 37 and 44ºC). Metabarcoding studies were then conducted through FLA 18S amplicons sequencing; am-plicon sequence variants (ASV) were extracted from each sample and taxonomy assigned against SILVA database using QIIME2 and SHAMAN pipelines. Vermamoeba were detected in DNA extracted directly from the soil, but to detect other FLA an amoe-bal enrichment step is necessary. The cultures were mainly enriched with Naegleria, Acanthamoeba and Vermamoeba, at 37ºC and 44ºC. High differences in FLA diversity were observed between the 27 sites. V. vermiformis was by far the most represented species of FLA, being detected throughout the islands while the genus Naegleria was mainly found in Basse-Terre. Nevertheless, putative pathogenic Nf was also sampled in Grande Terre and in Les Saintes Islands. Acanthamoeba were mainly found in areas where temperature is approx. 30ºC. FLA are an unrecognized risk and our results clearly show that FLA are widespread in Guadeloupe, posing a potential threat (direct or indirect) on human health in Guadeloupe. Similar studies could be performed in neighbouring Caribbean islands.