A comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) method has been developed for the formation and identification of unresolved complex mixtures (UCMs) in lacustrine biodegraded oils that with the same source rock, similar maturity, and increasing degradation rank from Nanxiang Basin, China. Normal alkanes, light hydrocarbons, isoprenoids, steranes, and terpanes are degraded gradually from oil B330 to oil G574. The compounds in biodegraded oil (oil G574) have fewer types, the polarity difference of compounds in different types is minor, and the relative content of individual compounds is similar. All the features make the compounds in biodegraded oil coelute in GC analysis and form the raised "baseline hump" named UCMs. By injecting standard materials and analyzing mass spectrums of target compounds, it is shown that cyclic alkanes with one to five rings are the major components of UCMs. Furthermore, UCMs were divided into six classes. Classes I and II, composed of alkyl-cyclohexanes, alkyl-naphthanes, and their isomers, are originated from the enrichment of hydrocarbons resistant to degradation in normal oils. Classes III ~ VI, composed of sesquiterpenoids, tricyclic terpanes, low molecular steranes, diasteranes, norhopanes, and their isomers, are probably from some newly formed compounds during the microbial transformation of oil.