Background: Fluoropyrimidines remain integral components of modern chemotherapy for solid tumors, and their toxicities can be reduced by pretreatment DPYD genotyping. Our main objective was to demonstrate the feasibility of implementing a hospital-wide pretreatment DPYD testing service based on the CPIC 2013 guideline on fluoropyrimidines and DPYD.Methods: We enrolled participants prior to planned fluoropyrimidine treatment as well as those who had experienced adverse events (AEs) after initiation of therapy, from December 1, 2013 to November 30, 2018. The patients tested pretreatment were analyzed as a prospective cohort to assess AEs within 90 days of fluoropyrimidine initiation and associated hospital cost. The primary outcome was the rate of severe global fluoropyrimidine-related toxicity in the pretreatment cohort (grade≥3, CTCAE v.4.0.3). Results: Of 1362 patients genotyped for DPYD within the study period 1041 were enrolled pretreatment and included in the primary analysis. The median age was 65 years (19-90), 57% male, 51% 5-FU, and 49% capecitabine. Dose reductions were recommended for 21 DPYD variant carriers who were detected pretreatment. There was no significant difference in the primary outcome between DPYD variant (29%) and wild type (18%) patients (Fisher’s exact test p = 0.25). Costs associated with ER visits and hospitalizations at our tertiary care centre were $1,268 (89-8,562) (Median (IQR)) and $2,961 (341-13,567) for DPYD variant (n = 4) and wild-type (n = 99) patients respectively. Post-AE genotyping (n = 70) found five DPYD variant patients; all experienced grade≥3 toxicity, costs were $15,825 (10,962-25,310), and one poor metabolizer died due to complications. Targeted next generation exome sequencing of DPYD wild-type patients who experienced severe AEs identified five potentially deleterious genetic variants in ABC efflux transporters. Conclusions: Pretreatment DPYD genotype guided dosing of fluorouracil and capecitabine is feasible and benefits patients, health care providers, and hospitals. Our data supports adoption of pretreatment DPYD genotyping as a standard of care.