Background: Vitamin D deficiency may be associated with an increased risk of asthma. Objective: We studied the association between 25-hydroxy (25-OH) vitamin D deficiency and asthma prevalence in two Peruvian populations close to the equator but with disparate degrees of urbanization. Methods: We conducted a population-based study in 1441 children in two communities in Peru, of which 1134 (79%) provided a blood sample for 25-OH vitamin D analysis. Results: In these 1134 children, mean age was 14.8 years; 52% were boys; asthma and atopy prevalence was 12% in Lima vs. 3% in Tumbes (P < 0.001) and 59% in Lima vs. 41% in Tumbes (P < 0.001), respectively; and, mean 25-OH vitamin D level was 20.8 ng/mL in Lima vs. 30.1 ng/mL in Tumbes (P < 0.001). Prevalence of 25-OH vitamin D deficiency (< 20 ng/mL) was 47% in Lima vs. 7% in Tumbes (P < 0.001). In multi-variable logistic regression, we found that lower 25-OH vitamin D levels were associated with an increased odds of asthma (OR = 1.7 per each 10 ng/mL decrease in 25-OH vitamin D levels, 95% CI 1.2-2.6; P < 0.01). In stratified analyses, the association between lower 25-OH vitamin D levels and asthma was limited to children with atopy (OR = 2.2, 95% CI 1.3-3.6) and not in those without atopy (OR = 0.9, 95% CI 0.5-2.0). We did not find associations between 25-OH vitamin D levels and other clinical biomarkers for asthma, including exhaled nitric oxide, total serum IgE and pulmonary function. Conclusion and Clinical Relevance: Both asthma and 25-OH vitamin D deficiency were common among children living in Lima (latitude = 12.0°S) but not among those in Tumbes (3.6°S). The relationship between 25-OH vitamin D deficiency and asthma was similar in both sites and was limited among children with atopy. Future supplementation trials may need to consider stratification by atopy at the time of design.