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An experimental investigation of optical diffraction in three-dimensional self-assembled photonic crystals is herein presented. The crystals are synthesized using polystyrene colloidal particles of four different sphere diameters and diffraction is studied under three different wavelength illuminations. The differences in the relative intensities of diffracted spots indicate the twin face-centered cubic lattice arrangement of the crystals. This is confirmed by monitoring the variation in intensity of diffracted beam with respect to the incident angles. Moreover, the diffraction angle versus the incidence angle plot provides us with an estimate of the colloidal sphere diameter.