We defined reliable and sensitive PCR assays to specifically detect trichothecene-producing Fusarium spp. in milling fractions and kernel tissues of naturally infected durum wheat. The assays were based on a combination of primers derived from the trichodiene synthase and the β-tubulin genes. The occurrence of toxigenic Fusarium spp. in semolina and wheat tissues (grain ends, crease, pericarp, aleurone layer, germ and albumen) was detected, even for a weakly contaminated wheat sample. We therefore demonstrated the penetration of toxigenic Fusarium spp. into the interior of durum wheat kernel for the Nefer variety and indicated that none of the tissue structures within the wheat kernel acted as an effective barrier to fungal invasion. Moreover, after inoculation by toxigenic Fusarium strains, semolina was shown to allow high yields of trichothecenes while bran was demonstrated to contain biochemical inhibitors able to significantly reduce trichothecenes production. These results will be useful for improving breeding strategy to control trichothecenes contamination of durum wheat kernels.