Background: Deleterious polymorphisms in gene-encoding DPD (DPYD) may result in the severe reduction of DPD enzymatic activity that causes life-threatening toxicities when the standard dose of fluorouracil is used. DPYD*2A (IVS14+1G > A) is the most common single-nucleotide polymorphism (SNP) associated with critical DPD deficiency. To enhance prevention of fluoropyrimidine toxicity, we assessed the potential clinical impact of additional DPYD polymorphisms. Methods: In 2011, we began screening DPYD*2A in patients candidate for fluoropyrimidine based-chemotherapy. We planned a case-control study with all cases of DPYD*2A wild type who developed CTC-NCI-V.3 toxicity ≥ G3 and with a cohort of patients who did not present severe toxicities (ratio 1:1.5). The two groups were matched for tumour site, staging (I-III vs IV) and patient’s age. Then we tested the additional SNPs (c.2846A > T, c.1679T > G, c.2194G > A) using Real Time PCR. Results: From 2011 to 2016 we screened 1,827 patients for DPD deficiency, of those 31 subjects (1.7%) showed DPYD*2A SNP. Complete clinical information was available only for 668 patients and of those, 146 (21.9%) developed severe toxicities (Case group). A control group was instead established with 220 patients who experienced no or mild toxicities. Fifty-three patients carried a variant in one of the additional SNPs: 35 subjects (66%) fell into the Case group and 18 (34%) into the Control group (OR 3.53, 95% IC 1.91-6.53. p < 0.0001). c.2194G > A was the most frequent SNP (12.5%, 46 of 366 pts) and showed a correlation with hematologic toxicity. In particular, neutropenia was observed in 50% of the patients carrying c.2194G > A (23 out of 46) vs 21% of patients c.2194 WT (67 out of 320) (OR = 3.75 95%IC 1.98-7.10; p < 0001). We confirmed that c.2826A > T (1.37%, 5 of 366 pts) was related to various toxicity (p = 0.0097) and c.1679T > G (0.55%, 2 of 366 pts) showed only gastrointestinal toxicity (p = 0.0027). Conclusions: Our data suggested that additional DPYD polymorphisms could enhance prevention of fluoropyrimidine toxicity. c.2194G > A is the most frequent polymorphism and it resulted associated with neutropenia.