In this study, durum wheat kernels harvested in three climatically different Italian cultivation areas (Emilia Romagna, Umbria and Sardinia) in 2015, were analyzed with a combination of different isolation methods to determine their fungal communities, with a focus on head blight (FHB) complex composition, and to detect fungal secondary metabolites in the grains. The genus was the main component of durum wheat mycobiota in all investigated regions, with the Central Italian cultivation area showing the highest incidence of this fungal genus and of its secondary metabolites. was the second most prevalent genus of the fungal community in all cultivation environments, even if regional differences in species composition were detected. In particular, Northern areas showed the highest incidence, followed by Central and then Southern cultivation areas. Focusing on the FHB complex, a predominance of , in particular in Northern and Central cultivation areas, was found. , in the analyzed year, was mainly detected in Emilia Romagna. Because of the highest incidence, durum wheat harvested in the Northern cultivation area showed the highest presence of secondary metabolites. These results show that durum wheat cultivated in Northern Italy may be subject to a higher FHB infection risk and to mycotoxins accumulation.