Wheat proteins are involved in respiratory allergy, contact allergy and food allergy. Wheat allergens involve in these pathologies are well-known. However, establishment of wheat allergy diagnostic can be sometimes difficult on account of the complex allergenic composition of skin prick test (SPT) solutions of wheat flour. Therefore, we have studied specific IgE reactivity from patient sera with wheat food allergy, and characterized allergenic composition of wheat SPT solutions by specific antibodies directed to wheat allergens. The results showed that 20 of the 25 sera analyzed contained specific IgE to at least one wheat protein fraction. Among positive sera, 75% have specific IgE to water/salt soluble fraction, 85% to native gluten fractions and 65% to wheat isolate fraction. The results showed also that SPT solutions of wheat flour contained major food allergens from each allergenic fraction. These results highlighted the importance of using fractions, which constitute the whole wheat allergenic pattern, during specific IgE reactivity analyses. Moreover, we have observed that wheat isolate extract (results of food industrial process) contained not only modified allergens (neo-allergens) involve of specific food allergy to wheat isolate but also some native allergens involve in wheat food allergy. Thus, these results showed the importance to use, for wheat in vivo diagnosis together wheat SPT solutions (gluten extract and wheat isolate) in order to differentiate wheat food allergy to specific wheat isolate allergy.