Although the quest to clarify the role of wild birds in the spread of the highly pathogenic H5N1 avian influenza virus (AIV) has yielded considerable data on AIVs in wild birds worldwide, information regarding the ecology and epidemiology of AIVs in African wild birds is still very limited. During AIV surveillance in Zambia (2008-2009), twelve viruses of distinct subtypes (H3N8, H4N6, H6N2, H9N1 and H11N9) were isolated from wild waterfowls. Phylogenetic analyses demonstrated that all the isolates were of the Eurasian lineage. While some genes were closely related to those of AIVs isolated from wild and domestic birds in South Africa, intimating the possible AIV exchange between wild birds and poultry in southern Africa, some gene segments were closely related to those of AIVs isolated in Europe and Asia, thus confirming the interregional AIV gene flow among these continents. Analysis of the deduced amino acid sequences of internal proteins revealed that several isolates harbored particular residues predominantly observed in human influenza viruses. Interestingly, the isolates having human-associated residues exhibited higher virus replication in lungs of infected mice and caused more morbidity as measured by weight loss than the isolate lacking such residues. Our study stresses the need for continued monitoring of AIVs in wild and domestic birds in southern Africa to better understand the emergence of strains with potential to infect mammals.