Seed-borne diseases of wheat such as Fusarium head blight (FHB), a fungal disease caused by several species of Fusarium, results in reduced yield and seed quality. The aim of this study was to identify the Fusarium species, the effect of Fusarium-infected seeds on germination and vigor indices and to determine the location of Fusarium spp. in seeds, as well as to investigate the pathogenicity and variability of aggressiveness of the isolates obtained from pre-basic seeds wheat fields in Iran. According to morphological and molecular characters, the species F. graminearum, F. culmorum, F. avenaceum and F. poae were identified. Among the isolates, F. graminearum was the predominant species with the highest frequency and relative density of 92.9% and 70.9%, respectively. We observed that germination and vigor indices were decreased due to increased Fusarium-infected seeds. Results indicated significant differences among cultivars and seed-borne Fusarium levels. While a higher infection level of Fusarium spp. most commonly occurred in the seed coat, only F. graminearum was observed in embryos. Our study about pathogenicity showed that 77.3% of the Fusarium spp. isolates were not pathogenic and 22.7% isolates of Fusarium spp. were pathogenic or weakly pathogenic. Our results indicated that variability in aggressiveness among isolates of a species and positive correlation may be determined by pathogenicity tests. This is the first time the location of Fusarium spp. in seeds has been identified. It is also the first time that Fusarium-infected seeds in pre-basic seeds wheat fields of Iran have been evaluated.