In this study, a total of 299 grain samples of wheat were collected from four production regions: the maize, sugar beet, potato and feed sectors of Slovakia. The samples were analyzed for deoxynivalenol (DON) content by using an enzyme-linked immunosorbent assay Ridascreen® Fast DON. Analysis of variance revealed a significant difference between years in DON contents (p < 0.027). The occurrence of samples with DON was 82.2% in 2010, with maximum DON content of 7.88 mg kg−1, and 70.7% in 2011, with maximum DON content of 2.12 mg·kg−1. The total mean DON content was 0.62 mg·kg−1; in the feed region 0.22 mg·kg−1; 0.63 mg·kg−1 in the maize region; 0.78 mg·kg−1 in the sugar beet region; 0.45 mg·kg−1 the potato region. The limit of 1.25 mg·kg−1 imposed by the European Union (EU) for DON content was exceeded in 13.7% of the studied samples. The average monthly rainfall for May to June played a critical role in DON content of wheat grains for maize and sugar beet producing regions. The present results indicate that DON content was at a high level in grains from wheat grown during 2010.