Cardiolipins (CL) are mitochondria specific lipids. They play a critical role in ATP synthesis mediated by oxidative phosphorylation. Abnormal CL distribution is associated with several disease states. MALDI-MS and MALDI-MS/MS were used to demonstrate in situ analysis and characterization of CL from tissue sections of organs containing high concentrations of mitochondria. Once the experimental parameters were established, a survey of CL distribution in heart, liver, kidney, leg muscle, and testis was undertaken. The major CL specie in the heart muscle, leg muscle, liver, and kidney is the (18:2)(4) CL, while liver and kidney also contain a minor specie, (18:2)(3)/(18:1) CL. The major CL specie in testis is the (16:0)(4) CL. The CL species distribution in various organs appeared to be in agreement with prior reports. Overall, proper matrix selection, tissue section handling, instrument tuning, and the inclusion of cesium ion in matrix ensured successful in situ MALDI-MS and MALDI-MS/MS analysis of CL. Upon modification and standardization, this method could be streamlined for rapid pathological diagnosis with short turnaround time in clinical settings.