Fractionation of petroleum during migration through sedimentary rock matrices has been observed across lengths of meters to kilometers. Selective adsorption of specific chemical moieties at mineral surfaces and/or the phase behavior of petroleum during pressure changes are typically invoked to explain this behavior. Given the current emphasis on unconventional (continuous) resources, there is a need to understand petroleum fractionation occurring during expulsion and migration at the nanometer to micron scale, due to the fine-grained nature of petroliferous mudrocks. Here organic matter compositional differences observed within kukersite petroleum source beds (containing acritarch Gloeocapsomorpha prisca) from the Ordovician Stonewall Formation are explored using a suite of optical and spectroscopic methods, most notably through a combined atomic force microscopy - infrared spectroscopy (AFM-IR) approach. The AFM-IR technique is capable of providing spatial resolutions approaching 50 nm and allows for an assessment of the molecular fingerprint of kukersite organic matter across transition zones from organic-rich ‘source’ layers into adjacent carbonate ‘reservoir’ layers ~150 μm away. Our results indicate that the composition of kukersite organic matter begins to vary immediately following expulsion from source layers, with loss of carbonyl groups and a concomitant increase in the CH3/CH2 ratio, indicating alkyl chain-length decrease, as migration distance increases. These chemical transitions correlate with fluorescence decrease, reflectance increase, and an increase in Raman proxies for aromaticity in the organic matter. These data are consistent with the retention of polar compounds onto mineral grains during expulsion and migration, and primacm-1ry cracking and bituminization of the Gloeocapsomorpha prisca kerogen, respectively.