Avian Influenza virus (AIV) H5N1 2.3.4.4b has emerged as a globally dominant clade. Unexpectedly, viruses from two genotypes within this clade have spilled over and spread in dairy cattle across multiple outbreaks, challenging existing assumptions about host range and zoonotic potential. To better understand spillover and adaptation potential, and to inform effective control strategies, it is essential to determine whether replication within a bovine host is a generalised feature of H5Nx viruses, specific to viruses of clade 2.3.4.4b, or more narrowly restricted to certain genotypes within this clade. We characterised the replication phenotypes of a broad panel of recombinant viruses bearing internal gene segments from 15 distinct clade 2.3.4.4b genotypes spanning over 60 years of evolution. Our data show that replicative fitness in bovine cells and ex vivo udder tissue is highly variable across clade 2.3.4.4b genotypes, limited in viruses predating the clade, and shaped by the internal viral gene cassette. Mutations in PB2 that emerged in bovine-derived B3.13 viruses are key determinants of adaptation, although their phenotypic effects are context-dependent. While recombinants bearing bovine-adapted internal segments replicated robustly, some avian-derived viruses also showed efficient replication in bovine cells and may be primed for spillover and further mammalian adaptation. Identifying these pre-adapted AIVs may improve zoonotic risk assessment beyond surveillance focused largely on known mammalian adaptation markers.