Fusarium head blight (FHB) is a global cereal disease caused by a complex of Fusarium species. In Europe, the main species responsible for FHB are F. graminearum, F. culmorum and F. poae. However, members of the F. tricinctum species complex (FTSC) have become increasingly important. FTSC fusaria can synthesize mycotoxins such as moniliformin (MON), enniatins (ENNs) and several other biologically active secondary metabolites that could compromise food quality. In this study, FTSC isolates primarily from Italian durum wheat and barley, together with individual strains from four non-graminaceous hosts, were collected to assess their genetic diversity and determine their potential to produce mycotoxins in vitro on rice cultures. A multilocus DNA sequence dataset (TEF1, RPB1 and RPB2) was constructed for 117 isolates from Italy and 6 from Iran to evaluate FTSC species diversity and their evolutionary relationships. Phylogenetic analyses revealed wide genetic diversity among Italian FTSC isolates. Among previously described FTSC species, F. avenaceum (FTSC 4) was the most common species in Italy (56/117 = 47.9%) while F. tricinctum (FTSC 3), and F. acuminatum (FTSC 2) accounted for 11.1% (13/117) and the 8.5% (10/117), respectively. The second most detected species was a new and unnamed Fusarium sp. (FTSC 12; 32/117 = 19%) resolved as the sister group of F. tricinctum. Collectively, these four phylospecies accounted for 111/117 = 94.9% of the Italian FTSC collection. However, we identified five other FTSC species at low frequencies, including F. iranicum (FTSC 6) and three newly discovered species (Fusarium spp. FTSC 13, 14, 15). Of the 59 FTSC isolates tested for mycotoxin production on rice cultures, 54 and 55 strains, respectively, were able to produce detectable levels of ENNs and MON. In addition, we confirmed that the ability to produce bioactive secondary metabolites such as chlamydosporol, acuminatopyrone, longiborneol, fungerin and butanolide is widespread across the FTSC. •Fusarium tricinctum species complex (FTSC) are important cereal pathogens worldwide.•Phylogenetic analyses revealed Italian FTSC isolates are genetically diverse.•Four of the nine FTSC species recovered in Italy represent novel phylospecies.•Fusarium avenaceum (FTSC 4) was the predominant species on Italian cereals.•FTSC isolates produced significant levels of moniliformin and enniatin mycotoxins.