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Agouti gene plays a critical role in the synthesis and distribution of coat color in domestic animals.In order to investigate the sequence structure characteristics of (Neovison vison) Agouti gene and it s mechanism to regulate the pelage color for fur animals, in the present study, the complete intron 2 nucleotide sequence of Agouti gene from the American short-haired black mink was obtained by PCR amplification and clone sequencing.The results showed that the obtained Agouti gene of black mink was 1335 bp in length, including complete intron 2 (1256 bp), partial exon 2(28 bp) and exon 3(51 bp), which had been submitted to the GenBank database and the accession number was KJ488543.The nucleotide composition for intron 2 sequence was 30.73%(A), 28.26%(T), 21.66%?, and 19.35%(G).TheA+T ratio(58.99%) was higher than that of G+C ratio(41.01 %).The sequence similarity of Agouti gene intron 2 between American short-haired black mink and coffee mink, red fox, dog, giant panda, domestic cat, horse, pig, alpaca, sheep, goat, cattle andrabbit were 99.8%,84.6%, 84.2%, 84.9%, 82.2%, 80.7%, 77.3%, 77.1%, 76.3%, 76.4%, 75.6%and 71.2%, respectively.The result of homology analysis showed that the genetic relationship between black mink and coffee mink was the highest, which was consistent with that they all belong to Mustela of the Mustelida animals in tranditional classification.Based on the sequence of Agouti gene intron 2, three phylogenetic trees were constructed for mink and other 11 species using Clustal X 1.83and MEGA 5.0 software by means of NJ, UPGMA and MP method, respectively.Evolutionary analysis revealed that black mink and coffee mink were sisters in their own clade, which was basically consistent with the taxonomy in the NationalCenter for Biotechnology Information.All above these findings would provide certain important biological informations to some extent for exploring the formation mechanism of different pelage colors and artificially cultivating a new coat color breed of mink.