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Abstract Background Infertility is very common condition and almost 50% of cases are due to male factors. Several genetic and environmental factors are responsible for the poor quality and reduced number of sperms in several cases of infertility. The present study was designed to investigate the association between semen parameters, homocysteine, and the risk of C677T polymorphism of MTHFR gene in infertile males of Iraqi population. Methods This Case–control study has been conducted from February 2019 to July 2021 at a molecular laboratory in the Anatomy and Histology Department/college of Medicine/University of Kufa/Najaf/Iraq. It was composed of 353 infertile male patients. They were divided into five groups: 90 azoospermic, 84 oligospermia, 64 asthenospermic, 50 oligoasthenospermic, and 65 teratospermic with an age range 20–46 years compared with 100 fertile males as control with age range 21–49 years. In order to detect homocysteine levels, we used Hcy ELISA Kit. C677T mutation of MTHFR gene was employed by PCR–RFLP technique. Results Our data revealed three genotypes of MTHFR C677T, 167 (47.3%) subjects had CC genotype, 116 (32.9%) subjects had CT genotype and 70 (21.1%) subjects had TT genotype. Furthermore, T allele was associated with higher risk of infertility in all patients groups for any genetic model. In total infertile subjects (codominant model: CT vs. CC, OR = 2.0, 95% C.I = 1.2–3.3, P = 0.011; TT vs. CC, OR = 4.8, 95% C.I = 3.3–8.2, P = 0.0003; dominant model: CT + TT vs. CC, OR = 2.8, 95% C.I = 1.7–4.5, P = 0.0001). Oligoasthenospermic patients associated with higher risk in CT heterozygous genotype (OR = 2.8, 95% C.I = 1.0–4.9, P = 0.03) and TT homozygous of mutant allele (OR = 6.3, 95% C.I = 1.9–9.2, P = 0.002). Homocystein level was elevated in all infertile groups when compared with control group (P |