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Mansonellosis is an undermapped insect-transmitted disease caused by filarial nematodes that are estimated to infect hundreds of millions of people globally. Despite their prevalence, there are many outstanding questions regarding the general biology and health impacts of the responsible parasites. Historical reports suggest that the Colombian Amazon is endemic for mansonellosis and may serve as an ideal location to pursue these questions in the backdrop of other endemic and emerging pathogens. We deployed molecular and classical diagnostic approaches to survey Mansonella prevalence among adults belonging to indigenous communities along the Amazon River and its tributaries near Leticia, Colombia. Deployment of a loop-mediated isothermal amplification (LAMP) assay on blood samples revealed an infection prevalence of ∼40% for Mansonella ozzardi . This assay identified significantly more infections than blood smear microscopy or LAMP assays performed using plasma, likely reflecting greater sensitivity and the ability to detect low microfilaremias or occult infections. Mansonella infection rates increased with age and were higher among males compared to females. Genomic analysis confirmed the presence of M. ozzardi that clusters closely with strains sequenced in neighboring countries. We successfully cryopreserved and revitalized M. ozzardi microfilariae, advancing the prospects of rearing infective larvae in controlled settings. These data suggest an underestimation of true mansonellosis prevalence, and we expect that these methods will help facilitate the study of mansonellosis in endemic and laboratory settings.