Dihydropyrimidine dehydrogenase (DPYD) is the rate-limiting step in fluoropyrimidines metabolism. Currently, genotype-guided fluoropyrimidine dosing is recommended for four DPYD single nucleotide variants (SNVs). However, the clinical impact of additional DPYD SNVs on fluoropyrimidine-related toxicity remains controversial. We assessed common DPYD SNVs c.85T>C, and c.496A>G which are often in linkage disequilibrium with c.1236G>A, a variant currently recommended for DPYD genotyping, in a retrospective cohort of cancer patients who had received fluoropyrimidines (N = 1371). When assessing individual SNVs, during the total chemotherapy treatment period, a significant increased risk of severe grade ≥ 3 toxicity was seen in carriers of c.496A>G (OR = 1.38, 95% CI 1.01-1.88, p = 0.0405) after adjusting for age, sex and treatment drug (capecitabine or 5-Fluorouracil). No association with fluoropyrimidine-related toxicity was seen in patients given standard dosing among those carrying one allele of DPYD c.1236G>A (OR = 1.19, 95% CI 0.59-2.27, p = 0.6147) or c.85T>C (OR = 1.04, 95% CI 0.80-1.62, p = 0.7536). Haplotype analysis confirmed a high linkage disequilibrium of these three variants. Toxicity was not significantly increased in haplotypes containing only one of c.85T>C or c.496A>G or c.1236G>A alleles. However, the haplotype containing both c.85T>C and c.496A>G alleles, which had a predicted frequency of 7.1%, was associated with an increased risk of fluoropyrimidine toxicity (OR = 1.57, 95% CI 1.15-2.13, p = 0.0041). This study suggests DPYD haplotype structure may help explain previous conflicting studies concerning the impact of these variants. Our findings suggest patients with both DPYD c.85T>C and c.496A>G variants have a significant increased risk for toxicity and may potentially benefit from genotype-guided fluoropyrimidine dosing.