It is known that pigs acted as "mixing vessels" for genesis of a new reassortant influenza strain responsible for pandemic H1N1 2009. However, the host factors driving the evolution of a reassorted virus in pigs to 'jump species' resulting in a human outbreak remain unclear. N-glycans derived from the porcine respiratory tract were enzymatically released, fluorescent labeled with 2-aminopyridine, separated according to charge, size and hydrophobicity, and structurally identified by a two-dimensional (size and hydrophobicity) HPLC mapping technique and MALDI-TOF mass spectrometry before and after exo-glycosidase digestion. We found a 3-, 5-, and 13-fold increases in NeuAc alpha 2-6, a preferable human influenza receptor, over NeuAc alpha 2-3, an avian influenza receptor, from upper and lower parts of the porcine trachea towards the porcine lung, a major target organ for swine virus replication. The large proportion of NeuAc alpha 2-6 may exert selective pressure for selection of influenza variants with altered receptor preference for this human-type alpha 2-6 receptor, a crucial first step for generating a human pandemic.