The first example of chromatographic separation based on isotopic chirality is shown. Diastereomers of methyl 3-phenyl-3-phenyl-d(5)-glycidate were separated by reversed-phase high-performance liquid chromatography based on the isotopic chirality provided by the presence of phenyl and phenyl-d(5) groups at one of the two chiral centers. Each of the two separated peaks was fractionated and subsequently separated into two peaks representing individual enantiomers having isotopic chirality by using a chiral stationary phase.