The aggressiveness of 32 Fusarium graminearum strains isolated from durum wheat cultivated in Northern-Central Italy was assessed. The chemotype (15-acetyldeoxynivalenol-, 3-acetyldeoxynivalenol- or nivalenol-producer) of each strain was characterized with multiplex PCR. Two aggressiveness assays (single floret inoculation and the Petri-dish test), which yielded three aggressiveness indices, were used. One strain from southern Italy (moderately aggressive) was added as the positive control to evaluate the accuracy of the aggressiveness quantification in both assays. The results showed that all 32 F. graminearum strains are pathogenic and a wide range of aggressiveness levels was observed. The positive control strain is pathogenic and showed moderately aggressive in both assays, thus indicated that our aggressiveness quantifications are valid. Six highly aggressive strains, representing all three chemotypes, were stable in the different aggressiveness assays. There was no significant difference in aggressiveness among the three different chemotypes (P = 0.909–0.972) determined by three aggressiveness indices. Highly significant correlations of AUDPC_standard to mean and terminal disease severity (r = 0.91, P < 0.001 in both cases) reveal the potential of using the Petri-dish test for selection of F. graminearum inocula with the proper and/or varying aggressiveness levels for breeding purposes.