Background There are several ecological studies, but few studies of the prevalence of SARS-COV-2 at high altitude. We aimed to estimate the population-based seroprevalence of SARS-COV-2 in three settings of Cusco at the end of the first wave among adults. Methods A population-based survey was conducted in September 2020, in three settings in the region of Cusco: (1) Cusco city at 3,300 meters above the sea level (m.a.s.l.), (2) the periphery of Cusco (Santiago, San Jerónimo, San Sebastián, and Wanchaq) at 3,300 m.a.s.l., and (3) Quillabamba city, located at 1,050 m.a.s.l. People aged [greater than or equal to] 18 years within a family unit were included. The diagnosis of SARS-CoV-2 infection was based on identifying anti- SARS-CoV-2 total antibodies (IgM and IgG) in serum using the Elecsys Anti-SARS-CoV-2 chemiluminescence test. Results We enrolled 1924 participants from 712 families. Of the total, 637 participants were anti-SARS-CoV-2 seropositive. Seroprevalence was 38.8% (95% CI [33.4%-44.9%]) in Cusco city, 34.9% (95% CI [30.4%-40.1%]) in the periphery of Cusco, and 20.3% (95% CI [16.2%-25.6%]) in Quillabamba. In 141 families (19.8%; 95% CI [17.0%-22.8%]) the whole members were positive to the test. Living with more than three persons in the same house, a positive COVID-19 case at home, and a member who died in the last five months were factors associated with SARS-COV-2 seropositivity. Dysgeusia/dysosmia was the symptom most associated with seropositivity (aPR = 2.74, 95% CI [2.41-3.12]); whereas always wearing a face shield (aPR = 0. 73; 95% CI [0.60-0.89]) or a facial mask (aPR = 0.76, 95% CI [0.63-0. 92) reduced that probability. Conclusions A great proportion of Cusco's city inhabitants presented anti-SARS-CoV-2 antibodies at the end of the first wave, with significant differences between settings. Wearing masks and face shields were associated with lower rate of seropositivity; however, efforts must be made to sustain them over time since there is still a high proportion of susceptible people.