Background: Prospective DPYD genotyping prevents severe fluoropyrimidine (FP)-induced toxicity by decreasing dosages in DPYD variant allele carriers. FP dosages in chemoradiation therapy (CRT) are lower than those in other FP-containing regimens. Pharmacogenetic guidelines do not distinguish between regimens, leaving physicians in doubt to apply dose reductions. Our aim was to investigate severe toxicity in DPYD variant allele carriers receiving CRT. Methods: Medical records of 828 patients who received FP-based CRT were reviewed from three centres. Severe (grade ≥III) toxicity in DPYD variant allele carriers receiving upfront FP dose reductions according to pharmacogenetic dosing guidelines and DPYD variant allele carriers not receiving FP dose reductions was compared with DPYD wild-type patients receiving standard dose of FPs in CRT. Results: DPYD variant allele carriers treated with standard dosages (N = 34) showed an increased risk of severe gastrointestinal (adjusted OR = 2.58, confidence interval [CI] = 1.02–6.53, P = 0.045) or severe haematological (adjusted OR = 4.19, CI = 1.32–13.25, P = 0.015) toxicity compared with wild-type patients (N = 771). DPYD variant allele carriers who received dose reductions (N = 22) showed a comparable frequency of severe gastrointestinal toxicity compared with wild-type patients, but more (not statistically significant) severe haematological toxicity. Hospitalisations for all DPYD variant allele carriers were comparable, independent of dose adjustments; however, the mean duration of hospitalisation was significantly shorter in the dose reduction group (P = 0.010). Conclusions: Standard FP dosages in CRT resulted in an increased risk of severe toxicity in DPYD variant allele carriers. We advise to apply FP dose reductions according to current guidelines in DPYD variant allele carriers starting CRT.