Head blight of wheat in the United States is caused primarily by the deoxynivalenol (DON)-producing chemotype of Fusarium graminearum. However, the discovery of the nivalenol (NIV) chemotype of F. graminearum in Louisiana and Arkansas necessitates having resistance in wheat to both chemotypes. The objectives of this research were to quantify resistance of selected winter wheat lines to initial infection and pathogen spread within spikes, to determine whether wheat lines selected for resistance to the DON chemotype also have resistance to the NIV chemotype, and to improve the methods for quantifying resistance to initial infection. A susceptible check (Coker 9835) and 15 winter wheat lines, which are adapted to the southeastern United States and possess diverse sources of head blight resistance, were evaluated for head blight resistance in a series of greenhouse and growth-chamber experiments. Significant levels of resistance to both initial infection and spread within a spike were found among the lines, and lines with resistance to isolates of the DON chemotype had even higher levels of resistance to isolates of the NIV chemotype. Quantifying resistance to initial infection was improved by standardizing the inoculum and environmental conditions. Additional information related to resistance to spread within a spike was obtained by calculating the area under the disease progress curve from 7 to 21 days after inoculation.