Background: TAS-102 is an oral combination of trifluridine (FTD) and tipiracil hydrochloride. Incorporation of tri-phosphorylated FTD into DNA is the main anti-tumor mechanism of action. We hypothesized that homologous recombination (HR) of DNA double-strand breaks (DSB) will modulate TAS-102 efficacy. We therefore tested whether genetic polymorphisms in the HR pathway are associated with clinical outcomes in patients with refractory metastatic colorectal cancer (mCRC) treated with TAS-102. Methods: We analyzed genomic DNA extracted from 104 blood samples of two different cohorts: an evaluation set of 52 patients receiving TAS-102 (median age 61 years, male 44%, median follow-up time 6.4 months); and a control set of 52 patients receiving regorafenib without history of TAS-102 treatment (median age 64 years, male 44%, all patients deceased). Ten single nucleotide polymorphisms (SNPs) of seven genes in HR pathway (ATM, BRCA1, BRCA2, XRCC3, FANCD2, H2AX, RAD51) were analyzed by PCR-based direct sequencing for association with progression-free survival (PFS) and overall survival (OS). Candidate SNPs were selected by their frequency and potential function. Results: In univariate analysis, patients carrying any A allele in XRCC3 rs861539 had significant longer PFS (3.8 vs. 2.3 months, HR 0.44, 95%CI: 0.21-0.92, p = 0.024) and OS (15.6 vs. 6.3 months, HR 0.25, 95%CI: 0.08-0.79, p = 0.012) than those with the G/G variant. Patients with any G allele in ATM rs609429 had a longer OS compared to those with the C/C variant (8.7 vs. 4.4 months, HR 0.37, 95%CI: 0.14-0.99, p = 0.022). In multivariate analysis, XRCC3 rs861539 showed marginal significance in PFS (p = 0.091) and OS (p = 0.056). ATM rs609429 remained significant for OS (p = 0.020). No significant differences were observed in the control group. Conclusions: Genetic variants in the HR pathway, ATM rs609429 and XRCC3 rs861539, may serve as predictive and prognostic markers in mCRC patients receiving TAS-102.