Circulating avian influenza viruses pose a significant threat, with human infections occurring infrequently but with potentially severe consequences. To examine the dynamics and locale of the adaptation process of avian influenza viruses when introduced to a mammalian host, we infected ferrets with H5N1 viruses. As expected, all ferrets infected with the human H5N1 isolate A/Vietnam/1203/2004 showed severe disease and virus replication outside the respiratory tract in multiple organs including the brain. In contrast infection of ferrets with the avian H5N1 virus A/Chicken/Laos/Xaythiani26/2006 showed a different collective pattern of infection; many ferrets developed and cleared a mild respiratory infection but a subset (25-50%), showed extended replication in the upper respiratory tract and developed infection in distal sites. Virus from these severely infected ferrets was commonly found in tissues that included liver and small intestine. In most instances the virus had acquired the common virulence substitution PB2 E627K but, in one case, a previously unidentified combination of two amino acid substitutions at PB2 S489P and NP V408I, which enhanced polymerase activity, was found. We noted that virus with high pathogenicity adaptations could be dominant in an extra-respiratory site without being equally represented in the nasal wash. Further ferret passage of these mutated viruses resulted in high pathogenicity in all ferrets. These findings illustrate the remarkable ability of avian influenza viruses that avoid clearance in the respiratory tract, to mutate towards a high pathogenicity phenotype during just a single passage in ferrets and also indicate a window of less than 5 days in which treatment may curtail systemic infection.