The analysis of triglyceride species by high performance liquid chromatography (HPLC) with a flame ionization detector (FID) and reversed-phase chromatography using chemically bonded octadecyl silane (ODS) Zorbax columns and gradient or isocratic solvent elution with methylene chloride/acetonitrile is described. Triglycerides containing acyl groups of critical pairs,trans and positional isomers, as well as mixtures of even and odd chain lengths are separated. Identification of triglycerides is made on the basis of retention times compared with equivalent and theoretical carbon numbers, and comparison with chromatograms of reference triglyceride mixtures. The methodology is demonstrated by fractionizing the triglycerides of olive oil under different chromatographic conditions using single and coupled conventional 250×4.6 mm columns and a short 80×6.2 mm column for fast separations.