The furin (FR) complex with each of four different sequences of hemagglutinin from the highly pathogenic H5N1 strains (HPH5), which were identified during the 2004-2010 influenza outbreaks in Thailand, were evaluated by molecular dynamics simulations, so as to compare the specificity and recognition of the enzyme-substrate binding. Relative to the conventional HPH5 inserted (H5Sq1, RERRRKKR), the S5-R or S6-R arginine residue is replaced by the smaller lysine in the H5Sq2 (RERKRKKR) and H5Sq3 (REKRRKKR) strains, respectively, whereas the S3-K lysine residue is deleted in H5Sq4 (RERRR-KR). The molecular dynamics results of the intermolecular interactions, in terms of hydrogen bonds and per-residue decomposition energy, between the substrate and furin revealed that the deletion of the positively charged amino acid at the S3 position in H5Sq4 leads to a notably weaker binding and specificity with the furin active site compared with that of FR-H5Sq1. A slight change in the substrate binding was found in the FR-H5Sq2 and FR-H5Sq3 complexes as a result of the replacement of the arginine with the shorter side-chained lysine (same positive charge). Altogether, the predicted binding free energy of the enzyme-substrate complexes was found to be in the following order: FR-H5Sq1 < FR-H5Sq2 ∼ FR-H5Sq3 << FR-H5Sq4.