The production of seeds with high genetic quality requires accurate estimates of the natural cross-pollination rate in cultivated Capsicum species. This is important in order to establish the most suitable isolation distance among fields of distinct genotypes, to minimize the risk of genetic contamination and to ensure that the agronomic and horticultural traits of an elite breeding material are preserved in a given seed lot. Cross-pollination studies in C. annuum are practically non-existent under Brazilian conditions. The objective of the present study was to estimate the natural rate of C. annuum cross-pollination in seed production fields established in the highland area of Central Brazil (“Cerrado”). A molecular marker (CA-Pun1), which allows the identification of cross-fertilization between C. annuum accessions with pungent and sweet fruits, was employed to identify hybrid seeds. This marker detects functional and non-functional alleles of an acyl-transferase (Pun1) gene, responsible to produce capsaicin (pungency) in placental tissue of C. annuum fruits. Seedlings of cultivar ‘Magali R’ (sweet fruits) were planted in the field at incremental distances from cultivar ‘BRS Garça’ (pungent fruits). Progenies obtained from ‘Magali R’ plants were genotyped with marker CA-Pun1. Approximately 10.8% of the fruits displayed at least one seed originated from cross-pollination. The overall natural cross-pollination rate in C. annuum was estimated at 1.2%, according to the percentage of hybrid seeds detected by molecular analysis. No significant differences were detected in the rate of cross-pollination among plants for distances of up to 7.2 m.