The organic matter occluded in the silica of fossil diatom frustules is thought to be protected from diagenesis and used for paleoceanographic reconstructions. However, the location of the organic matter within the frustule has hitherto not been identified. Here, we combined high spatial resolution imaging by nanoSIMS and Raman micro-spectroscopy to identify where the organic material is retained in cleaned fossil diatom frustules. NanoSIMS imaging revealed that organic signals were present throughout the frustule but in higher concentrations at the pore walls. Raman measurements confirmed the heterogenous presence of organics but could not, because of lower spatial resolution, resolve the spatial patterns observed by nanoSIMS.