The fluorescence color and mol. signature of oil inclusions can provide precious information on the thermal maturity of inclusion oils and the fluid migration history of basins. Here we show how mol. mass spectrometric analyses in combination with fluorescence microscopy can be used to reveal heterogeneities in the chem. compositions of oil inclusions within a micrometer sized quartz crystal. We used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to extract and directly analyze the mol. content of two yellow and two white-blue fluorescing oil inclusions in a single quartz crystal from the Barrandian Basin (Czech Republic). The cyclic ion species detected primarily in the yellow fluorescing oil inclusions likely originate from steranes, hopanes, and other tricyclic and tetracyclic terpanes, whereas these are less abundant in the white-blue fluorescing oil inclusions. Depth profiles of the fluid inclusions indicate that the white-blue fluorescing oil inclusions are emptied faster in the ToF-SIMS instrument, presumably due to a higher content of more volatile components than in the yellow fluorescing oil inclusions. The phenanthrene to dibenzothiophene ratio derived from ToF-SIMS of the four inclusion oils from the Barrandian Basin indicates marine clastic sediments as the source rocks for all the inclusion oils. The phenanthrene to alkylphenanthrene ratio derived from ToF-SIMS indicates no major difference in thermal maturity of the oil in the white-blue and yellow fluorescing oil inclusions. Instead, variation in the chem. content of the trapped oil induced by trapping fractionation may be the key control.