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 F. 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 F. 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 F. graminearum population.