Clade 2.2 Eurasian lineage H5N1 highly pathogenic avian influenza viruses (HPAIV), were first detected in Qinghai Lake, China in 2005, and subsequently spread through Asia, Europe and Africa. Importantly, these viruses carried a lysine at position 627 of the PB2 protein (PB2 627K), a known mammalian adaptation motif. Previous avian influenza isolates have carried glutamic acid in this position (PB2 627E), commonly described to restrict virus polymerase function in the mammalian host. We sought to examine the effect of PB2 627K on viral maintenance in the avian reservoir. Reverse genetics viruses were engineered to contain converse PB2 627K/E mutations in a Eurasian H5N1 virus (A/turkey/Turkey/5/2005; Ty/05) and, for comparison, a historical pre-Asian HPAI H5N1 virus that naturally bears PB2 627E (A/turkey/England/50-92/1991; 50-92). 50-92 PB2 627K was genetically unstable during virus propagation resulting in reversion to PB2 627E or the accumulation of additional mutations PB2 628R and/or a synonymous mutation PB2 a1869g. Intriguingly PB2 628R and/or a1869g appeared to improve the genetic stability of 50-92 PB2 627K. However, the replication of 50-92 PB2 627K in conjunction with these stabilizing mutations was significantly restricted in experimentally infected chickens where reversion to PB2 627E occured. In contrast, no significant effects on viral fitness was observed for Ty/05 PB2 627E or K in in vitro or in vivo experiments. Our observations suggest PB2 627K is supported in Eurasian lineage viruses; in contrast PB2 627K carries a significant fitness cost in the historical pre-Asian 50-92 virus.