Aggressiveness toward wheat of 56 new Fusarium accessions, obtained from random naturally infected grain samples of bread wheat, durum wheat and triticale collected across eight year/location combinations from Romania, phenotypically and by molecular tools was investigated. Molecular techniques allowed identification of Fusarium species and the analysis of polymorphism within fungal isolates. A large overall variation of aggressiveness, phenotypically expressed as reduction of coleoptiles length (ranging from 2.1 to 48.2% of control), in two independent trials performed in seedling stage, on average over three varieties, was registered. Field point inoculations at anthesis of 90 Fusarium isolate x wheat varieties combinations also revealed variability of several components of aggressiveness in adult stage: severity (14.4-64.8%), AUDPC (104.9-527.1) and FDK (8.1-43.7%), respectively. The presence of TRI5 gene involved in DON biosynthesis was detected in most isolates, allowing the assumption of their high toxigenic potential associated with the other aggressiveness traits. Both DON chemotypes, 3ADON and 15ADON, were identified in Fusarium populations from Albota, Brasov, Livada and Simnic. Similarity between records obtained in seedling and adult stage for the most aggressive of Fusarium isolates, suggests that phenotypic selection, in conjunction with molecular tools, could be a reliable method to select the appropriate pathogen strains for breeding of resistance.