Background: Fluoropyrimidines are widely used in the treatment of various solid tumors. One-third of cancer patients develop severe treatment-related toxicity. A part of fluoropyrimidine-related toxicity arises due to impaired activity of dihydropyrimidine dehydrogenase (DPD) caused by genetic variants in the DPYD gene. Objective: This study aims to determine the frequency of DPYD mutations among patients with adverse effects of 5-fluorouracil therapy. Materials and Methods: This cross-sectional study recruited 46 patients with 5-FU adverse effects. All demographic, clinical, and laboratory findings were recorded. Molecular testing was conducted to detect the DPYD mutation using the PGX-5FU StripAssay® kit, which identifies the IVS14 + 1G>A DPYD variant (DPYD*2A), the most common DPYD mutation. Results: The majority of the 46 recruited Iraqi Arab patients were females (71.74%), and the most common age groups were 41–50 and 51–60 years (26.08%) each. The most common cancer was breast cancer (45.65%). Poorly differentiated tumors were the most common grade (60.86%), and stage 3 was the most common tumor stage (47.82%). 5-FU was used as a single agent in 19.57%. The most common adverse effects reported were alopecia, vomiting, anemia, fatigue, diarrhea, and neutropenia. Molecular testing revealed that all patients had the wild-type DPYD IVS14 + 1G>A genotype. Conclusions: This study supports the evidence that routine testing for DPYP mutation in patients receiving 5-FU is not cost-effective and that selected patients can be tested if they show toxic reactions suggestive of DPD deficiency. Testing for four common variants of the DPYD gene can achieve better results.