The water buffalo has a seasonal reproductive pattern with reduced sexual activity during the longer photoperiod. The goal of this study was to identify the single nucleotide polymorphism (SNP) of melatonin receptor 1A (MTNR1A) gene in Iraqi buffalo cows and 3D structure of its protein and phylogeny with other sequences around the world. The 824 bp fragment of exon II of the MTNR1 A gene was amplified from 190 buffalo cows (4-5 years old) genomic DNA belonging to local breeders in Al-Chibayish Marshes, Southern Iraq. Amplified PCR products underwent custom sequencing at the two ends (5′ and 3′ ends). Five separate polymorphism sites, the 1st included 52 animals with 19 mutations (12 missense), the 2nd included 39 animals with 18 mutations (11 missense), the 3rd included 35 animals with 18 mutations (12 missense), the 4th included 32 animals with 18 mutations (12 missense) and the 5th included 32 animals with 14 mutations (8 missense). These polymorphic sites with accession numbers LC565046, LC565047, LC565709, LC565710 and LC565711 respectively were registered in gene bank. The phylogenetic tree reveals that in some of the Iraqi buffalo, the sequences of MTNR1A gene has identical to the Italian buffalo (GU817415), and the Brazilian buffalo (JN689386). Data revealed a marked difference in the fifth polymorphism sites' 3D protein structure because of the mutations. In conclusion, as a result of mutations, the gene MTNR1A in Iraqi buffalo has polymorphisms; these polymorphisms may be linked to gene function, Therefore, further studies are needed to connect the polymorphisms of this gene with the productive and reproductive traits.