The manner in which deoxynivalenol (DON) and nivalenol (NIV) accumulation progresses in wheat grain infected with Fusarium graminearum and the influence of the time of infection on the accumulation of toxins were investigated. Four cultivars were tested in a greenhouse environment, where the plants were spray inoculated at three different stages with a mixture of DON and NIV chemotypes of F. graminearum. The results indicate that high levels of DON and NIV can be produced beyond 20 days after anthesis (DAA), even by early infection. The results of field experiments performed on seven cultivars, where inoculation was conducted using colonized maize kernel inoculum, were consistent with the greenhouse results. In addition, in the greenhouse experiments, late infection, at least as late as 20 DAA, caused grain contamination with these toxins even without clear disease symptoms on the spike. These results indicate the importance of the late stage in grain development in DON and NIV contamination, suggesting that control strategies that cover the late as well as the early stage of grain development should be established to effectively reduce the risk of these toxins' contaminating wheat.