Synchrotron microfocus small angle X-ray scattering was used to investigate the nanostructure and microscopic variation of eggshells. It uses a microbeam allowing the ability to probe interactions between the organic and inorganic components at nanometer level and is ideal for mapping over small areas to obtain a detailed analysis of structural variations. Thin sections of eggshells were scanned from the shell membrane (inner) to the cuticle (outer) surface. The data collected was used to produce two-dimensional maps showing microscopic changes within the different layers of the eggshell. The structural alterations apparently could have implications at the macroscopic level of the resulting eggshell. As the organic matrix is embedded within the eggshell this may contribute to the variations observed in calcite crystal form and texture. Structural information obtained about a biomaterial at different length scales is important in relating the structure to its functional properties. This knowledge and the principles behind the formation of biomaterials could be used in the attempt of bioengineering new systems.