Health benefits of high molecular weight (MW) β-glucans are well documented. Therefore, understanding and controlling depolymerization of β-glucan in baked products, would increase the effectiveness of β-glucan to confer health benefits. In this study we demonstrated that endogenous β-glucanase in wheat kernels are responsible for the depolymerization of β-glucans. A protocol was developed based on the Megzayme procedure to detect low levels of β-glucanase activity in wheat flour. This was confirmed by using HPLC-Calcofluor detection to monitor molecular weight changes. The distribution of β-glucanase in wheat kernels was also investigated. The effect of genotype, location, planting season and environmental factors on the level of endogenous β-glucanase in selected wheat cultivars was investigated using different wheat varieties planted under different condition and different seasons. Furthermore, kinetics of β-glucan depolymerization by endogenous glucanase in two dough systems with different moisture content was investigated. The results demonstrated that enzymes with β-glucanase activity are concentrated primarily in the outer layer of wheat kernels. Also genotype, environmental conditions and agronomic practice all had significant effects on the β-glucanase activity in wheat flours and poor harvesting conditions can significantly increase β-glucanase activity level in wheat. The kinetics results demonstrated that moisture content, incubation time and levels of endogenous β-glucanase activity of the system had significant impact on the final MW of β-glucan in the dough. Among all factors investigated, moisture content had the greatest impact. This study presents opportunities for industry to fortify baked products with high molecular weight β-glucan.