Strongyloides stercoralis affects 30-100 million people worldwide. The burden is underestimated because of the paucity of studies, limited geographical areas surveyed, and poor quality of diagnostic tests. This study aimed at determining the epidemiology of strongyloidiasis using sensitive microscopy testing in rural populations living at different altitudes in Cusco, Peru. Data were collected from subjects aged > 3 years living in Quellouno (elevation 2,600 ft) and Limatambo (elevation 8,379 ft) districts. Subjects provided one fresh stool sample and answer a standardized questionnaire. Fresh stool was tested on site using the Baermann's test and agar plate culture. Formalin-preserved stool was tested by rapid sedimentation. Eighty percent (585/715) of eligible subjects consented to participate; after excluding subjects with missing data, 65% (462/715) were included. Fifty-five percentage were female; the median age was 33 years (interquartile range 13-52), and 72% had government health insurance. Half had intestinal parasites, and Strongyloides was the most common (24.5%) followed by Giardia (15.5%), Blastocystis (14.9%), and hookworm (11.5%). The agar plate culture detected more cases of Strongyloides than Baermann's or sedimentation tests. Strongyloides infection was more common at low altitude (26.4%) than at high altitude (18.6%), but the difference was not statistically significant (P = 0.08). Older age, walking barefoot, bathing in rivers/streams, and using municipal sewage were associated with strongyloidiasis. Strongyloides was the most prevalent parasite in the areas studied and was associated with demographic, socioeconomic, and sanitary factors.