Background Melioidosis is an emerging infectious disease caused by the soil-dwelling bacterium Burkholderia pseudomallei that affects both humans and animals. It is endemic in South and Southeast Asia, and northern Australia, causing an estimated 165,000 human cases annually worldwide. Human cases have been reported in the French West Indies (Martinique and Guadeloupe) since the 1990s. Conversely, no human cases have been reported in French Guiana, a French territory in South America. Our study aimed to investigate whether B. pseudomallei is locally established in Guadeloupe and French Guiana. We assessed animal exposure by serology and examined the presence of B. pseudomallei in the environment of seropositive animals. Methodology/Principal findings Blood samples were collected from domestic animals in two goat farms in Les Saintes, Guadeloupe (n=31), and in 56 farms in French Guiana (n=670) and tested by ELISA. Serological follow up was performed on selected farms. Soil, water and goat rectal swabs were collected and analysed by culture and PCR. In French Guiana, the highest prevalence rates were observed in equids (24%) and cattle (16%), while in Les Saintes, a prevalence of 39% was observed in goats. The longitudinal study in Les Saintes revealed consistent high seropositivity in goats. A B. pseudomallei strain was isolated from the soil from one of the farms and detected in goat rectal swabs from the other farm. Conclusions/Significance Our environmental investigation prompted by the serologic data confirms the presence of B. pseudomallei in Les Saintes, consistent with documented human cases of melioidosis on this island. In French Guiana, our serologic results call for environmental surveys and a re-evaluation of human infections with melioidosis-like symptoms. The approach developed in this study may help to identify high-risk areas that warrant further investigation. Author summary Burkholderia pseudomallei , an environmental bacterium, is the causative agent of melioidosis in humans and animals. If the disease has been historically reported to be endemic in South Asia and northern Australia, recent studies reveal its presence outside of these territories, both in the environment and among patients who have not travelled to endemic areas. Furthermore, the projected increase in extreme climatic events in the near future could increase the prevalence of the disease as well as cause its emergence in new territories. For these reasons, it is important to identify new areas at risk. Our study aimed to investigate the presence of the pathogen in French West Indies. We combined surveys in domestic animals (cattle, goats, horses, sheep, and pigs) and in the environment. The identification of seropositive animals without clinical signs, together with the isolation of B. pseudomallei in the environment of a goat farm in Guadeloupe, underscores the importance of including melioidosis in animal surveillance programs. The use of serologic methods can help identify animal exposure to the pathogen, thereby helping to identify areas where the pathogen may be present in the environment.