The development of electrospray ionization mass spectrometry has been critical for the analyses of lipidomes from subcellular organelles. The myocardial nuclear lipidome likely has a key role in the molecular regulation of gene expression. In fact, recent studies have suggested that specific phospholipid classes bind and regulate specific transcription factors. The dynamic regulation of the myocardial nuclear lipidome may be critical in mediating long-term pathological responses to stresses such as ischemia, tachycardia, and hypertension. In this brief review, the preparation of myocardial nuclei is discussed, and the resulting nuclear lipidome from rat and rabbit are shown as examples. The rabbit myocardial nuclear lipidome contains relatively more plasmenylcholine/phosphatidylcholine molecular species in comparison to that ratio observed in the rat myocardial nuclear lipidome. The composition of the rat myocardial nuclear choline glycerophospholipid pool was relatively enriched with molecular species containing arachidonic acid and docosahexaenoic acid in comparison to that in the rabbit myocardial nuclear choline glycerophospholipid pool. While the ethanolamine glycerophospholipids of the rabbit myocardial nuclei are enriched with arachidonic acid and plasmalogens, the ethanolamine glycerophospholipid profile from rat myocardial nuclei show less plasmalogen and more species containing docosahexaenoic acid. Last, significant differences in the ethanolamine glycerophospholipid molecular species were observed in the rabbit heart lipidomes from the nucleus and the mitochondria. Quantitation of these lipid species in hearts subjected to pathophysiological stresses may provide important information on the role of the myocardial nuclear lipidome on long-term cardiac cell function.