The current work aims to evaluate the association between genetic mutations in thymidylate synthetase (TYMS gene in exon1 and partial regions of promotor and intron 1 [877 bp, 657,220–658,096 bp]) and the therapeutic outcomes for rheumatoid arthritis (RA) Iraqi patients. An observational cross-sectional study involving 95 RA patients with established RA patients based on their methotrexate treatment responsiveness. Genetic sequencing of the TYMS gene was performed for all patients according to the instruction manuals of the sequencing company (Macrogen Inc. Geumchen, South Korea). Four polymorphisms were identified by sequencing 95 randomly selected patients in the noncoding region of TYMS. Three of these polymorphisms were found in the NCBI database’s dbSNP (rs2853741, rs2606241, and rs2853742 SNPs), and one SNP polymorphism is novel (657334). The CTAT (657334, rs2853741, rs2606241, and rs2853742 SNPs) haplotype was significantly associated with responder with odd ratio, 95% confidence interval: 0.506, 0.281–0.912 (P value = .022). In contrast, the other haplotypes were not associated with MTX responsiveness. In the multivariate analysis, after adjusting to the effect of age, sex, smoking, and disease duration, the TC_rs2853741 genotype was associated with non-responders (P value = .030). In contrast, the AC_rs260641 genotype, after adjusting to the effect of age, sex, and smoking, was associated with non-responders (P value = .035). Genetic polymorphism of the TYMS gene, especially in TC_rs2853741 and AC_rs260641, predicts non-responder to MTX treatment in RA, while the presence of the CTAT haplotype predicts a good response to MTX treatment.