Viral evolution facilitates the emergence of viruses with unpredictable impacts on human health. We investigated the intra-host variations of receptor-binding domain, RBD, in HA gene of the avian H5N1 viruses obtained from 2004 and 2005 epidemics. The result showed that the mutation frequency of RBD ranged from 0.3-0.6 %. This mutation generated one consensus and several minor populations. The consensus population of 2004 epidemic was transmitted into the 2005 outbreak with increasing frequency, 39% to 45%, respectively. MD simulation was applied to predict the significance of variants. Results revealed that the consensus sequence (E218K/V248I) interacted unstably with the SAα2,6Gal. While K140R/E218K/V248I and Y191C/E218K/V248I sequences decreased HA binding capacity to α2,3-linked SA, they were shown to bind α2,6-linked SA with increased affinity. Moreover, 140 and 191 substitutions are positive-selection sites. Our data suggested that K140R and Y191C mutations may represent a step toward human adaptation of the avian H5N1 virus.