Genetic polymorphisms in drug metabolizing enzymes and drug transporters may affect irinotecan toxicity. Although genetic polymorphisms have been shown to influence the irinotecan toxicity, data are limited in Thai population. Thus, the aim of this study was to assess the allele and genotype frequencies and the relationship between CYP3A4/5, DPYD, UGT1A1, ABCB1, and ABCC2 genetic variations and irinotecan-induced toxicity in Thai colorectal cancer patients. One hundred and thirty-two patients were genotyped, and the effect of genetic variations on irinotecan-induced toxicity was assessed in 66 patients who received irinotecan-based chemotherapy. Allele frequencies of ABCB1 c.1236C > T, ABCB1 c.3435C > T, ABCC2 c.3972C > T, ABCG2 c.421C > A, CYP3A4*1B, CYP3A4*18, CYP3A5*3, DPYD*5, UGT1A1*28, and UGT1A1*6 were 0.67, 0.43, 0.23, 0.27, 0.01, 0.02, 0.64, 0.19, 0.16, and 0.09, respectively. DPYD*2A and DPYD c.1774C > T variants were not detected in our study population. The ABCC2 c.3972C > T was significantly associated with grade 1-4 neutropenia (P < 0.012) at the first cycle. Patients carrying both UGT1A1*28 and *6 were significantly associated with severe neutropenia at the first (P < 0.001) and second (P = 0.017) cycles. In addition, patients carrying UG1A1*28 and *6 had significantly lower absolute neutrophil count (ANC) nadir at first (P < 0.001) and second (P = 0.001) cycles. This finding suggests that UGT1A1*28, *6, and ABCC2 c.3972C > T might be an important predictor for irinotecan-induced severe neutropenia.