Alternaria is a saprophytic and opportunistic fungus with a worldwide distribution that can affect the quality of various agricultural products, such as fruits, cereals, and pseudocereals. This research was carried out to investigate the population of this genus associated with quinoa cultivation in plots located in the Boyacá department (Colombia), the country’s third-largest quinoa-producing department. The present study found 17 Alternaria isolates, of which 13 were identified as A. alternata and 4 as A. scrophulariae (formerly A. conjuncta) employed molecular markers of internal transcribed spacer (ITS) region and translation elongation factor 1α (TEF-1α). In the pathogenicity test under greenhouse conditions, all the Alternaria isolates showed some degree of pathogenicity on Piartal quinoa cultivar plants although no significant differences were found in isolates. The severity indices ranged from 2 to 5, and the percentage of affected leaves per plant ranged between 15% and 40%. This fungus affected the foliar tissue of quinoa, resulting in chlorotic and necrotic spots, symptoms that can generate a reduction in the quality and productivity of crops. This is the first time that the pathogenicity of Alternaria spp. in the Piartal variety has been described and the first report of this genera in quinoa crops of Colombia.