One hundred and six influenza A virus isolates were compared phylogenetically using gene segment 8 which encodes the two non-structural (NS) proteins, NS1 and NS2. The isolates separated into two previously described subtypes, A and B. For non-structural gene subtype A viruses, five subdivisions were distinguished in the nucleotide phylogenetic tree including a human-swine group, American avian-equine group, Eurasian avian-swine group, a unique gull isolate group, and a group with a single member, A/Equine/Prague/56. At the nucleotide sequence level the B subtype was subdivided into two groups, including the American avian group and Eurasian avian-equine group. In the analysis of the NS1 and NS2 predicted amino acid sequence, American and Eurasian influenza isolates became less distinguishable. Size variability of the NS1 protein was observed, especially in the human and swine isolates. The previously reported deletion in A/Turkey/Oregon/71 NS1 gene, which caused a truncated NS1 protein, was contrasted with a full length NS1 gene cloned from an earlier A/Turkey/Oregon/71 stock. All avian origin influenza viruses had a putative NS1 sequence length of 230 aa, with two exceptions, A/CK/Pennsylvania/1370/83 and A/CK/Pennsylvania/21525/83, with an NS1 protein of 217 aa.