Tumor Necrosis Factor-α (TNFα) is a cytokine signaling protein which has an important role in the immune system regulation and affect female reproductive performance. The objective of this study was investigation the TNFα gene polymorphism and its potential effects in female buffaloes fertility. The DNA was extracted from the blood of 81 buffalo females and a 592 bp fragment contains the full coding region of TNFα-exon 4 was amplified by PCR which subsequently treated with RsaI restriction enzyme. The PCR-RFLP pattern showed that all the animals had fixed CC genotype and T allele was not detected. Sequencing of amplified fragment (GenBank accession No. KY885010) flowed by sequence alignment with GenBank database revealed that the river buffalo target sequence was homologues to cattle than goat, sheep, human and mouse on DNA and amino acids levels. Comparison of TNFα amplicon with homologues Bubalus bubalis records in the GenBank detected 3 SNPs in exon 4. Two of these SNPs were synonymous while the third located in the 3` UTR. Different effects of some of discovered SNPs on RNA cis-regulatory elements and hn mRNA and mature mRNA secondary structures were predicted.