Deoxynivalenol (DON) is a mycotoxin produced by Fusarium graminearum and its intake represents severe risk to human and animal health. The objective of this study was to evaluate levels of DON in wheat (Triticum aestivum L.) flour from two representative locations of south Brazil. Experiments were carried out in Pato Branco (Paraná) and Coxilha (Rio Grande do Sul) in a randomized complete block design with three replicates. Levels of DON were measured by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). This mycotoxin was detected in 97% of samples, ranging from 200 to 4140 μg kg-1. Even though, FHB epidemics were slight lower in the growing season that the study was performed. Only 17% of samples presented DON beyond of the maximum allowed by the Brazilian Health Surveillance Agency up to the year 2012; even though, FHB epidemics were slight low in the growing season that the study was performed. According to our knowledge, this is the first report showing genetic variability of Brazilian cultivars to DON contamination and some genotypes have potential to be exploited as a source of low accumulation of this toxin.