Durum wheat production in Italy is of great importance from an economic point of view. In Italy, Fusarium graminearum is the main fusarium head blight (FHB) causal agent in wheat, reducing both grain quality and yield. F. graminearum produces trichothecene mycotoxins such as deoxynivalenol (DON) and nivalenol (NIV) that are toxic for humans and animals. A population of F. graminearum sensu stricto strains was collected from symptomatic durum wheat heads and grains from several naturally infected fields in different Italian regions such as Emilia Romagna, Lazio, Marche, Piemonte, Sardegna, Umbria and Veneto. A multiplex PCR in the Tri12 region of the trichothecene gene cluster was used to assign a strain to one of trichothecene chemotype profiles: NIV, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON). In order to evaluate possible mycotoxin contamination risks it is very important to know which chemotype is the prevalent in a F. graminearum population. Our research showed that the15ADON chemotype was the most frequent. This work, performed for the first time in Italy, allowed us to better understand the distribution of the F. graminearum chemotypes.