Fusarium graminearum and Fusarium culmorum are among the major causal agents of Fusarium head blight, which reduces both crop yield and grain quality in wheat worldwide. The present study was conducted with 57 isolates collected from 23 different locations across four provinces in the 2011/2012 growing season. Out of the 57 Fusarium isolates, 32 isolates were identified as F. graminearum and 25 isolates were identified as F. culmorum. Both pathogens are of particular importance, since they produce several mycotoxins. Among these, deoxynivalenol (DON) and nivalenol (NIV) are well known for their toxicity towards human and animal health. Genetic chemotyping of F. graminearum and F. culmorum species indicated that both DON and NIV chemotypes were present in the surveyed area. Of the 32 F. graminearum isolates, the primer sets Tri13DON and Tri13NIV identified 87.5% as DON chemotypes and 12.5% as NIV chemotypes. Similarly, the 25 F. culmorum isolates displayed 88% DON and 12% NIV chemotypes. In addition, DON acetylated derivatives, 3-acetyldeoxynivalenol (3-AcDON) and 15-AcDON, were identified by polymerase chain reaction based methods. It was determined that 15-AcDON sub-chemotype was dominant in F. graminearum populations, whereas 3-AcDON was dominant in F. culmorum populations. This is the first report demonstrating the presence of F. graminearum and F. culmorum isolates and the distribution of 3-AcDON and 15-AcDON chemotypes in both Fusarium species in wheat fields of eastern Mediterranean region of Turkey.