Abstract: The separation of diastereoisomers of acyclic isoprenoid alkanes on HP-5 columns was firstly reported by gas chromatography and mass spectrometry(GC-MS).The biomarkers of saturated hydrocarbons from coal samples of the Junggar Basin and crude oils of Gudao area were determined,and iC₁₈(2,6,10-trimethyl pentadecane),Pr(2,6,10,14-tetramethyl pentadecane),Ph(2,6,10,14-tetramethyl hexadecane) and their diastereoisomers were identified from coal samples and crude oils.The effects of type and length of chromatographic column and temperature programming on the separation of diastereoisomers of acyclic isoprenoid alkanes were investigated.The most favorable separation conditions were concluded.The diastereoisomers were separated from nor-pristane,Pr and Ph,and the maximal resolution factors of Pr and Ph are 0.7,0.9,respectively.