From 2014 to 2015, three cases of highly pathogenic avian influenza infection occurred in zoo-housed north-east China tigers (Panthera tigris ssp.altaica) and four tigers died of respiratory distress in succession in Yunnan Province, China. We isolated and characterized three highly pathogenic avian influenza A(H5N1) viruses from these tigers. Phylogenetic analysis indicated that A/tiger /Yunnan /tig1404 /2014(H5N1) belongs to the provisional subclade 2.3.4.4e which were novel reassortant influenza A (H5N1) viruses with six internal genes from avian influenza A (H5N2) viruses. The HA gene of the isolated A/tiger /Yunnan /tig1412 /2014(H5N1) virus belongs to the subclade 2.3.2.1b. The isolated A/tiger /Yunnan /tig1508/2015 (H5N1) virus was a novel reassortant influenza A (H5N1) virus with three internal genes (PB2, PB1 and M) from H9N2 virus and belongs to the subclade 2.3.2.1c.