The fungal disease Fusarium head blight affects cereal grains and can produce mycotoxins, like the water-soluble deoxynivalenol (DON). Wheat wet milling process begins with ground endosperm obtained by dry milling and ends with the separation of starch from gluten. Research was conducted on hard red spring wheat and durum wheat samples naturally contaminated with DON. The fate of DON in wheat dry milled fractionations (farina/semolina, shorts, and bran) during wet milling was investigated. Three wet milling processes were evaluated. DON levels were assessed by GC-ECD. Results showed that DON was present in all dry milled fractions. DON concentration in farina and semolina exceeded the safety threshold for human consumption. After wet milling farina and semolina, nearly all the DON was found in the water-soluble fraction, regardless the wet milling process. A negligible level of DON was found in the gluten extracted from HRSW with Martin wet milling process.