The recent report of the first fatality associated with infection by influenza virus H5N1 clade 2.3.4.4b, identified as genotype D1.1, which is distinct from the B3.13 genotype, has sparked fears of a potential human pandemic. However, the genetic relationships between B3.13 and D1.1, as well as their origins, host adaptability, and antiviral resistance, remain poorly understood. Here we conducted a comprehensive phylogenetic and comparative analysis of H5N1 clade 2.3.4.4b across multiple species, in order to identify the molecular characteristics and frequency of resistance mutations in these two genotypes, elucidate their evolutionary trajectories, and assess their implications for public health. Our results demonstrate that B3.13 exhibits mammalian adaptability, while D1.1 retains avian adaptability. Importantly, both genotypes display limited occurrences of human-like signatures, which can help alleviate public anxiety. Additionally, the emergence of the resistance mutations in the clade 2.3.4.4b on the binding sites of antivirals calls for the development of multi-target antiviral strategies to mitigate the risk of resistant strain reassortment.