The outermost layer of the skin, the stratum corneum, offers a protective barrier. In atopic eczema (AE) this skin barrier is impaired. Although AE is associated with filaggrin mutations, the reason for the impaired skin barrier function is inconclusive. Lipids in the stratum corneum (ceramides, fatty acids and cholesterol) are crucial for a proper skin barrier function, but their role in relation to atopic eczema is indistinct. In this study a comprehensive analysis of ceramide composition and lipid organisation in stratum corneum is performed. The ceramide composition is examined by mass spectrometry, while the lipid organization is monitored by Infrared spectroscopy and X-ray diffraction. Our studies show a change in the ceramide composition in stratum corneum of AE patients compared to healthy subjects. This concerns the ceramide subclasses as well as the chain length distribution in each of these ceramide subclasses. These changes in ceramide composition correlate excellent with an aberrant lipid organization. The observed lipid changes are not dependent on filaggrin mutations. We demonstrate changes in ceramide composition and lipid organization in the skin barrier of patients with AE. We foresee that these new insights will play an important role in future studies on therapy and prevention of AE.