Fusarium graminearum (Fg) is a devastating, agricultural pathogen which causes significant losses to cereal crops worldwide because it negatively impacts grain yield as well as grain quality by production of carcinogenic mycotoxins. Chemical fungicides are currently used to control this pathogen, but resistance is now common globally which is limiting their sustainable use. Thus there is an urgent need for new fungicides with different mechanisms of action. Chitin synthases (CHS) are excellent targets for new antifungal drugs, because chitin is essential to the integrity of the fungal cell wall and thus the survival and virulence of fungal cells. Furthermore, this polysaccharide not made by plants or mammals and thus this target is specific for fungi. No CHS inhibitors have been successfully developed for control of human or agricultural fungal pathogens. One reason for this, is the paucity of biochemical information on these important enzymes because they are embedded in the plasma membrane and cannot be purified in the quantities needed for biochemical analysis. This is further complicated by the presence of several chitin synthase genes in each fungal species. The aim of this study is to identify the full complement of chitin synthases in Fg and to recombinantly express them in yeast for biochemical characterisation. Eight potential CHS genes were identified in Fg using a combination of bioinformatics, functional complementation and microscopy. Two of these genes complemented a chs double knockout in S. cerevisiae and were taken forward for biochemical characterisation. The FgCHS3b and FgCHS4 proteins were successfully expressed as C-terminal GFP-tagged fusions; a challenging prerequisite for enabling purification and biochemical characterisation of these Fg CHS enzymes for the first time.