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We report a grazing incidence x-ray diffraction (GIXD) investigation of the surface lipid layer of the pre-ocular tear film. For the first time we demonstrate the existence of 2D order over a wide range of surface pressures in this system, with typical spicing of 3.75 Å and 4.16 Å independent of the monolayer surface pressure. Analogous lipid ordering is also found in an artificial lipid mixture of the major lipid components of the tear film, suggesting that the 2D ordering is set by generic lipid–lipid interactions. Fluorescence microscopy of the natural and artificial tear film mixture reveals the co-existence of a dilute and a much more condensed phase in the amphiphilic lipid matrix over the pressure range of 15–45 mN/m investigated by GIXD, plus an additional structure due to the much more hydrophobic part of the mixture. This evidence supports the previous hypothesis that tear film has a layered structure.