Background FP pharmacodynamics potentially depends on polymorphisms of genes related to its catabolism, anabolism, folate pathways, targets and transporters. EMA, CPIC and DPWG recommend DPYD testing (consensual variants *2A/*13/D949V/HapB3 of the DPYD gene coding for dihydropyrimidine dehydrogenase (DPD)) before starting FP chemotherapy. The main objective of this study is to identify a multigenic signature improving current recommendation to identify patients at risk of severe FP-induced toxicities. We herein present genetic results of DPYD sequencing. Methods The study included 3,807 Caucasian colorectal cancer patients from 8 studies (4 prospective cohorts, 4 trials) previously selected according to PRISMA Guidelines. All received 5FU- or capecitabine-based treatment without FP preemptive dose adaptation based on DPD status. NGS sequencing of germline DNA was performed by the Integragen company on a NovaSeq6000™ using a capture approach (Twist Bioscience®). In silico predictions were based on CADD, UMD-Predictor and Human Splicing Finder systems. Results Sequencing fulfilled quality control criteria for 3,471 DNA samples. DPYD sequencing identified 138 coding and 24,738 intronic variants, including 35 and 14,682 new ones, respectively. 35.1% of patients carried at least one known coding variant (4.9% with 2) other than the 4 consensual ones (prevalence 6.2 %). In silico analyses predicted 41 coding variants as deleterious and 6 as probably deleterious, while 4 intronic variants (at least one carried by 1.6% of patients) were predicted to impact splicing. Among the 51 deleterious/probably deleterious variants, 9 were novel polymorphisms, each identified in one patient (heterozygous): 5 deleterious missenses (L176P, C671R, R783H, A818T and C953S), one nonsense (L165*), one probably deleterious missense (V637M), and 2 intronic variants disrupting a donor site (c.850+5G>A) or a branch point (c.40-23C>T). Conclusions This is the largest series of patients with full DPYD sequencing reported so far. Less than 0.25% of patients carried a new deleterious DPYD coding variant and 1.6% carried a potentially functional intronic variant. Meta-analysis of their impact on toxicity is ongoing.