The highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b has rapidly disseminated globally, with mammalian infections reported in multiple species. Recent evidence of mammal-to-mammal transmission has heightened concerns about the virus’s potential adaptation to mammals. The polymerase basic 2 (PB2) protein E627K mutation appears to be of key importance for mammalian adaptation. We isolated an HPAI H5N1 clade 2.3.4.4b virus from wild birds in Korea with 96% E and 4% K at amino acid position 627 of PB2. To investigate the genomic characteristics of this clade regarding mammalian adaptation, we studied the replication and transmission of the H5N1 virus in mice. Two experiments with different challenge-to-contact ratios were conducted to assess transmission dynamics and mutation development. In experiment 1, a 4:1 challenge-to-contact ratio resulted in 100% transmission among direct-contact mice, with all mice succumbing to the infection. In experiment 2, a 1:1 ratio yielded 50% transmission, with all challenged mice also succumbing. High viral loads were observed in the lungs and brains in both experiments, with viral titers increasing over time. Notably, the PB2-E627K variant, initially present at 4% in the virus stock, was selected and reached near-fixation (~?100%) in the lungs and brains by 6 days post-challenge and was subsequently transmitted. No other mammalian-adaptive mutations were identified, emphasizing the pivotal role of PB2-E627K in early stages of mammalian adaptation. These findings highlight the need for continuous genomic monitoring to detect mammalian adaptation markers and assess interspecies transmission risks.