Fusarium graminearum of the 15-acetyl-deoxynivalenol (15-ADON) chemotype is the main cause of Fusarium head blight (FHB) of wheat in southern Brazil. However, 3-ADON and nivalenol (NIV) chemotypes have been found in other members of the species complex causing FHB in wheat. To improve our understanding of the pathogen biology and ecology, we assessed a range of fitness-related traits in a sample of 30 strains representatives of 15-ADON (F. graminearum), 3-ADON (F. cortaderiae and F. austroamericanum), and NIV (F. meridionale and F. cortaderiae). These included perithecia formation on three cereal-based substrates, mycelial growth at two suboptimal temperatures, sporulation and germination, pathogenicity toward a susceptible and a moderately resistant cultivar, and sensitivity to tebuconazole. The most important trait favoring F. graminearum was a two times higher sexual fertility (>40% perithecial production index [PPI]) than the other species (<30% PPI); PPI varied among substrates (maize > rice > wheat). In addition, sensitivity to tebuconazole appeared lower in F. graminearum, which had the only strain with effective fungicide concentration to reduce 50% of mycelial growth >1 ppm. In the pathogenicity assays, the deoxynivalenol producers were generally more aggressive (1.5 to 2× higher final severity) toward the two cultivars, with 3-ADON or 15-ADON leading to higher area under the severity curve than the NIV strains in the susceptible and moderately resistant cultivars, respectively. There was significant variation among strains of the same species with regards asexual fertility (mycelial growth, macroconidia production, and germination), which suggested a strain- rather than a species-specific difference. These results contribute new knowledge to improve our understanding of the pathogen-related traits that may explain the dominance of certain members of the species complex in specific wheat agroecosystems.