Members of the Fusarium graminearum species complex (FGSC) infect several cereal crops, including wheat, maize and sorghum, to cause diseases such as Fusarium head blight (FHB), Gibberella ear rot (GER) and grain mold (GM), respectively. These diseases reduce grain yield and quality, which result in food security and safety concerns as infected grain is often contaminated with mycotoxins. In 2011, an extensive survey in South Africa uncovered six of the 15 known FGSC members. This study provided the first investigation into the prevalence, geographic distribution and host range of these members in the country, and revealed evidence of host-specificity within the FGSC. The accurate identification of morphologically indistinguishable isolates found in South Africa is necessary to separate FGSC members, while host-specificity is an important concept when developing disease control strategies, as it affects the application of crop rotation systems. Therefore, a study was undertaken to i) evaluate matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) and polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) as potential methods for the identification of South African FGSC members; ii) determine the composition of FGSC members in fields cultivated under a wheat and maize crop rotation system in South Africa; iii) determine the mycotoxin chemotype of these FGSC members; and iv) determine host-specificity of FGSC members on wheat, maize and sorghum. Different MALDI-TOF MS spectra were obtained for the protein profiles of F. graminearum s.s., F. meridionale and F. boothii. PCR-RFLP profiles following restriction digests of the translocation elongation factor α-1 (TEF) and histone 3 (H3) gene regions distinguished four FGSC members from one another. A multiplex chemotype PCR revealed that wheat isolates belong almost exclusively to the 15-ADON chemotype, with only a single NIV chemotype isolate. In maize, 139 isolates comprised of a 15-ADON chemotype and six isolates with a NIV chemotype were identified for the first time. No 3-ADON chemotype isolates were identified in this study. Host-specificity of FGSC members was not observed, as all isolates resulted in significant disease development. However, F. graminearum s.s. was significantly more virulent on wheat and F. boothii on maize. This is consistent with the hypothesis that F. boothii is at a selective advantage to cause disease on maize grain.