A 2024 human HPAI H5N1 case in British Columbia showed mixed viral populations containing HA-190D (28%) and HA-226H (35%) variants in tracheal aspirate sequencing. In this study, solid-phase binding assay and molecular docking were used to evaluate the contribution of HA-E190D/Q226H mutations to the viral receptor profiles. Our results showed that HA-E190D marginally reduced sialic acid α2,3 receptors´ affinity, while HA-Q226H impaired both sialic acid α2,3 and α2,6 receptors´ binding. These results demonstrate that neither mutation strengthens viral binding to human-type receptors, indicating that such substitutions are unlikely to heighten the public health threat posed by the virus for now.