Cardiolipin (CL) is a class of phospholipid tightly associated with the mitochondria functions and a prime target of oxidative stress. Peroxidation of CL dissociates its bound cytochrome C, a phenomenon that reflects oxidative stress sustained by the organ and a trigger for the intrinsic apoptotic pathway. However, CL distribution in normal organ tissues has yet to be documented. Fresh rat organs were snap-frozen, cut into cryosections that were subsequently desalted with ammonium acetate solution, and vacuum-dried. CL distribution in situ was determined using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique on sections sublimed with 2,5-dihydroxybenzoic acid. CL images in rat cardiac ventricular section showed a homogeneous distribution of a single m/z 1447.9 ion species that was confirmed as the (18:2)4 CL by tandem mass spectrometry. The presence of low abundant (18:2)3(18:1) CL with the bulk (18:2)4 CL in quadriceps femoris rendered the muscle CL exhibiting a slightly deviated isotopic pattern from that of cardiac muscle. In rat liver, MALDI-MSI unveiled three CL-containing mass ranges, each with a unique in situ distribution pattern. Co-registration of the CL ion images with its stained liver section image further revealed the association of CLs in each mass range with the functional zones in the liver parenchyma and suggests the participation of in situ CLs with localized hepatic functions such as oxidation, conjugation, and detoxification. The advances in CL imaging offer an approach with molecular accuracy to reveal potentially dysregulated metabolic machineries in acute and chronic diseased states.