The comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) has been used to characterize a crude oil and a source rock extract sample. During the process, a series of pairwise components between monocyclic alkanes and mono-aromatics have been discovered. After tentative assignments of decahydronaphthalene isomers, a series of alkyl decalin isomers have been synthesized and used for identification and validation of these petroleum compounds. From both the MS and chromatography information, these pairwise compounds were identified as 2-alkyl-decahydronaphthalenes and 1-alkyl-decahydronaphthalenes. The polarity of 1-alkyl-decahydronaphthalenes was stronger. Their long chain alkyl substituent groups may be due to bacterial transformation or different oil cracking events. This systematic profiling of alkyl-decahydronaphthalene isomers provides further understanding and recognition of these potential petroleum biomarkers.