Management of Fusarium Head Blight (FHB), caused by Fusarium graminearum sensu stricto and other members of the F. graminearum species complex (FGSC), is challenging because of its complex epidemiology and the strong influence of environmental factors on disease outcomes and toxin accumulation. There is a high degree of genotypic and phenotypic variation among pathogen species and strains. Current FHB models and treatments do not account for pathogen diversity, so it is difficult to predict what will happen if a new variant is introduced, or if changes in the environment favor one genotype over another. To improve management of FHB, we aim to (1) identify novel genetic markers associated with fitness and pathogenicity, and (2) incorporate these markers in multi-locus genotyping assays to monitor and predict population shifts. Although they are homothallic, outcrossing occurs between and within species. Controlled crosses are challenging because outcrossed perithecia must be differentiated from selfed ones. We produced deletion mutants of the MAT1-1-1 and MAT1-2-1 (MAT) genes and screened them to identify appropriate test maters with normal fitness- and pathogenicity-related phenotypes. The deletion strains engage only in heterothallic mating, solving the problem of identifying outcrossed perithecia. Many strains, especially the MAT1-2-1 deletions, were significantly reduced in pathogenicity or fitness compared with the wild type (WT). However, two highly female-fertile MAT1-1-1 strains did not differ from WT, and these were used in test matings with two MAT1-2-1 deletions varying in colony morphology and pathogenicity, and with WT strains including the PH-1 progenitor, another strain of F. graminearum (Gz3639), and F. meridionale. Antibiotic resistance, MAT alleles, and fertility had expected 1-1 segregation patterns in the crosses with F. graminearum WT strains, while segregation patterns related to colony morphology were complex and indicated absence of linkage to the MAT deletions. The interspecific cross resulted in non-Mendelian segregation patterns. Analyses involving additional molecular markers and pathogenicity-related phenotypes are continuing. However, results so far suggest that the MAT1-1-1 deletion strains will be suitable as test mates for crosses with WT FGSC strains, facilitating our efforts to identify molecular markers linked to fitness, pathogenicity, and toxigenicity-associated phenotypes. Acknowledgements: This material is based upon work supported by the U.S. Department of Agriculture.  This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.