Aim: Head and neck cancer is identified by diverse group of malignant tumors that can develop in or all around the mouth, throat, nose and sinuses. It is a rather general multifactorial malignancy. The aim of the present study was to investigate the association of MTR (A2756G); MTRR (A66G); MTHFD1 (G1958) and RFC-1(G80A) gene polymorphisms in head and neck squamous cell carcinoma (HNSCC) patients and controls in North Coastal Andhra Pradesh. Materials and methods: A total of 220 samples (110 HNSCC patients and 110 controls) were included in the present study and genotyping was accomplished by using PCR - RFLP technique. Data was analyzed by SPSS 19 software. Results: The chi square p values revealed that MTR (A2756G) and RFC-1(G80A) gene polymorphisms has shows association with HNSCC and MTRR (A66G) and MTHFD1 (G1958)gene polymorphisms does not shows association with HNSCC. For MTR (A2756G) polymorphism the odds ratio p values of genotypes AG and GG were statistically significant, whereas AA genotype was found to be statistically insignificant. For MTRR (A66G) polymorphism the odds ratio p values of genotypes AA, AG and GG were found to be statistically insignificant. For MTHFD1 (G1958) polymorphism the odds ratio p values of genotypes GG, AG and AA were found to be statistically insignificant. For RFC-1(G80A) polymorphism the odds ratio p values of genotypes AG and GG were statistically significant, whereas AA genotype was found to be statistically insignificant. Conclusions: In conclusion, the MTR (A2756G) and RFC-1(G80A) gene polymorphisms shows association with HNSCC, whereas MTRR (A66G) and MTHFD1 (G1958) gene polymorphisms does not shows association with HNSCC.