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Background: The spread of the coronavirus disease 2019 (COVID-19) in Peru has been reported at the regional level, few studies have evaluated its spread at the provincial level, in which the mechanisms could be different. Methods: We conducted an analytical, cross-sectional, multistage observational population study to assess the seroprevalence of SARS-COV-2 at the provincial and urban/rural levels in a high-altitude setting. The sampling unit was the household, including a randomly selected family member. Sampling was performed using a data collection sheet on clinical and epidemiological variables. Chemiluminescence tests were used to detect total anti-SARS-COV-2 antibodies (IgG and IgM simultaneously). The percentages were adjusted to the sampling design. Results: The overall prevalence in the region of Cusco was 25.9%, with considerably different prevalence between the 13 provinces (from 15.9% in Acomayo to 40.1% in Canchis) and between rural (21.1%) and urban (31.7%) areas. In multivariable model, living in a rural area was a protective factor (adjusted prevalence ratio [aPR], 0.68; 95% confidence interval [CI], 0.61–0.76). Conclusions: Geographic diversity and population density determine different prevalence rates, typically lower in rural areas, possibly due to natural social distancing or limited interaction with people at risk.