Engineering compositional changes in oilseeds is typically accomplished by introducing new enzymatic step(s) and/or by blocking or enhancing an existing enzymatic step(s) in a seed-specific manner. However, in practice, the amounts of lipid species that accumulate in seeds are often different from what one would predict from enzyme expression levels, and these incongruences may be rooted in an incomplete understanding of the regulation of seed lipid metabolism at the cellular/tissue level. Here we show by mass spectrometry imaging approaches that triacylglycerols and their phospholipid precursors are distributed differently within cotyledons and the hypocotyl/radicle axis in embryos of the oilseed crop Camelina sativa, indicating tissue-specific heterogeneity in triacylglycerol metabolism. Phosphatidylcholines and triacylglycerols enriched in linoleic acid (C18:2) were preferentially localized to the axis tissues, whereas lipid classes enriched in gadoleic acid (C20:1) were preferentially localized to the cotyledons. Manipulation of seed lipid compositions by heterologous over-expression of an acyl–acyl carrier protein thioesterase, or by suppression of fatty acid desaturases and elongases, resulted in new overall seed storage lipid compositions with altered patterns of distribution of phospholipid and triacylglycerol in transgenic embryos. Our results reveal previously unknown differences in acyl lipid distribution in Camelina embryos, and suggest that this spatial heterogeneity may or may not be able to be changed effectively in transgenic seeds depending upon the targeted enzyme(s)/pathway(s). Further, these studies point to the importance of resolving the location of metabolites in addition to their quantities within plant tissues.