To elucidate the characteristics of variation and the genetic evolution of non-structural protein (NS1, NS2) genes related to avian influenza subtype H5N1 viruses isolated from the boundary region of Yunnan province.Swab samples were collected from foreign poultry and wild birds in the boundary regions of Yunnan province and screened by H5/N1 subtype-specific multiplex RT-PCR. The NS segment of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis on those available NS1, NS2 genes were performed with sequences of the known reference strains.71 positive samples were identified from 1240 samples, with the positive rate as 5.72%. Fourteen different NS segment sequences were obtained from 30 representative positive samples and could be divided into 3 distinct clades or sub-clades (I-1, I-2 and II), by phylogenetic analysis. The NS1/NS2 genes and Hemagglutinin (HA) genes of H5N1 viruses from the boundary regions of Yunnan province showed different relationships regarding the characteristics on genetic evolution. The substitution or mutation of key amino acids sites had been noticed in the nuclear location signal domains, effect domain, and other pathogenicity markers.NS genes of H5N1 subtype viruses in boundary region of Yunnan province showed genetic divergence and the virus of clade I-2 and II had become dominant epidemic strains in this region since 2010.