BACKGROUND AND PURPOSE: Relationships between gene change and virulence for hemagglutinin (HA) subtypes of avian influenza virus remain inconclusive. In this study, sequences of these nearly identical virus strains were obtained in order to elucidate the relationship between molecular determinants and virulence. METHODS: Two strains, with different virulence, of an H6N1 avian influenza virus were isolated from an infected chicken flock. Complete 8-gene fragments from the 2 strains were cloned and sequenced. Putative amino acid sequences were compared. RESULTS: Comparisons of the sequences from the 2 strains showed 0.65%, 0.79%, 0.28%, 1.23%, 0.80%, 0.20%, 0.43%, and 0.83% differences in PB2, PB1, PA, HA, NP, neuraminidase (NA), NS1 and NS2 proteins, respectively. The M1, M2, and PB1-F2 protein sequences from the strains were identical. The HA cleavage site of both strains contained a single R, despite their difference in virulence. Thus, the difference in virulence might be due to sequences other than the HA cleavage site. Most of the changes were in the HA2 part. The sequence immediately after the HA cleavage site was GILG in the non-virulent strain and GIFG in the virulent strain. The change from E to G at position 106 in the HA, near the receptor binding site, might influence the virulence. Other sequence changes likely to influence virulence were from K to R at position 291 (K291R) in NP protein and from P to T at position 101 (P101T) in NA protein. CONCLUSION: The amino acid changes identified in this study may be important in the virulence of influenza viruses.