Fusarium graminearum, one of the causal agents of fusarium head blight (FHB) of wheat in the UK, can be broadly divided into two chemotypes based on the production of the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). DON-producing isolates can be further distinguished on the basis of the predominant acetyl DON derivative that they produce; 3-acetyl DON (3-AcDON) or 15-acetyl DON (15-AcDON). Functional Tri13 and Tri7 are required for the production of NIV and 4-acetyl NIV, respectively, whereas, in isolates that produce only DON and its acetylated derivatives, these genes are nonfunctional or absent. Infections caused by E graminearum are becoming more frequent in the UK; however, it is unknown whether these represent more than one chemotype. In this study, polymerasae chain reaction (PCR) assays specific for functional and nonfunctional/deleted versions of Tri7 and Tri13 were used to characterize 101 single-spore isolates of Fusarium graminearum as DON or NIV chemotypes. Primer sets developed to Tri3 were used to classify DON chemotypes further by the acetyl derivative produced (3-AcDON or 15-AcDON). Isolates were collected from 65 fields located around England and Wales between 1997 and 2002. All three chemotypes were identified from the E graminearum population of England and Wales, with 15-AcDON chemotypes predominating overall. All isolates characterized as 3- or 15-AcDON chemotypes had nonfunctional versions of both genes. Where multiple isolates were collected from a field, mixed-chemotype populations were identified. Variation in the number of 11-bp repeats in Tri7 was detected among 3- and 15-AcDON chemotypes. Seventy-two of the 76 DON chemotypes (95%) were classified as 15-AcDON producers and the remaining four isolates (5%) as 3-AcDON producers. In all four isolates with a 3-AcDON chemotype, Tri7 was deleted from the trichothecene gene cluster. There was no evidence of regional variation between 3-AcDON, 15-AcDON or NIV chemotypes within the E graminearum population.