Chicken Mx protein confers resistance against viral infections including avian influenza virus. Mx protein is one of the strong antiviral proteins induced by interferon system. Different alleles of chicken Mx gene have been shown to vary in their antiviral activity against influenza virus. A non-synonymous mutation at position 2032 of chicken Mx gene responsible for variation of amino acid (Ser to Asn) in Mx protein is responsible for negative to positive antiviral activity of Mx protein. In the present study it was analyzed that SNP at G2032A in chicken Mx gene in two Indian native chicken breeds, Aseel, Kadaknath and compared with White Leghorn layer. A total of 90 samples, including 50 of White Leghorn and 20 each from native chicken breed were screened for SNP at position 2032 of Mx gene by using real time multiplex allele specific PCR. The resistant allele (A) frequency is higher than susceptible allele in all three chicken breeds investigated. Among the breeds, the resistant allele frequency (A) is higher in White Leghorn (0.95) followed by Aseel (0.75) and Kadaknath (0.625). Homozygous to resistant allele (AA) is the major genotype in White Leghorn whereas heterozygous to both alleles is the major genotype in Kadaknath. Variations in the gene frequency might have emerged from varying degree of selection pressure the different breeds had undergone. In future, the resistant genotype could be selected in modern breeding programs to improve the genetic status for disease resistance against viral infections