Three positional isomers of diglucosyl-cyclomaltohexaose (diglucosyl-cG₆) were chemically synthesized via 6¹, 6²-, 6¹, 6³-, and 6¹, 6⁴-di-O-(tert-butyldimethylsilyl)-cG₆s (1, 2, and 3) prepared regiospecifically. Glucosylation of bis(2, 3-di-O-acetyl)tetrakis(2, 3, 6-tri-O-acetyl)-cG₆s obtained from the three regioisomeric compounds 1, 2, and 3 with 2, 3, 4, 6-tetra-O-benzyl-1-O-trichloroacetimidoyl-α-D-glucopyranose, followed by debenzylaiton and then deacetylation, afforded 6¹, 6²-, 6¹, 6³-, and 6¹, 6⁴-di-O-(α-D-glucopyranosyl)-cG₆s (10, 11, and 12) together with configurational isomers. The desired compounds 10, 11, and 12 containing two (1→6)-α-linkages were isolated from the mixtures of their configurational isomers by high performance liquid chromatography. The three diglucosyl-cG₆s synthesized chemically were used as authentic samples to identify the components in a mixture of diglucosyl-cG₆s produced by an enzymatic process.