The regulation of lipid synthesis in oil seeds is still not fully understood. Oilseed rape is the third most productive vegetable oil crop on the global market. Therefore, increasing our understanding of lipid accumulation in oilseed rape seeds is of great economic, as well as intellectual, importance. Matrix-assisted laser/desorption ionisation - mass spectrometry imaging (MALDI-MSI) is a technique that allows the mapping of metabolites directly onto intact biological tissues, giving a spatial context to metabolism. We have used MADLI-MSI to study the spatial distribution of two major lipid species, triacylglycerols (TAGs) and phosphatidylcholines (PCs). A dramatic, heterogeneous landscape of molecular species was revealed, demonstrating significantly different lipid compositions between the various seed tissues. The embryonic axis was particularly enriched in lipid species containing palmitate, while the seed coat/aleurone layer accumulated vaccenic, linoleic and α-linoleic acids. Furthermore, the lipid composition of the inner and outer cotyledons differed to each other, a remarkable discovery given the supposed identical functionality of these two tissues. TAG and PC molecular species distribution was analysed through a developmental time series covering early seed lipid accumulation to the end of lipid accumulation. The spatial patterning of lipid molecular species did not vary much during seed development, although there were exceptions. Data gathered using MALDI-MSI was verified through gas chromatography analysis of dissected seeds. The distinct lipid distribution profiles observed implies differential regulation of lipid metabolism between the different seed tissues. Further understanding of this differential regulation will enhance efforts to improve oilseed rape productivity and quality.