Pyrolysis-gas chromatography-mass spectrometry (pyr-GC-MS) is emerging as a promising alternative for the detection and quantification of microplastic pollution. For a robust quantification it is essential to improve our understanding of interferences in the pyrolysis of microplastics. Here we investigate the effects of different soil matrices, mainly differing by their organic carbon content (Corg, 1.0–13.6%), and of the polymer molecular weight (Mw) on the pyr-GC-MS analysis of polystyrene (PS) microplastics. In addition, we evaluated the effectiveness of adding poly(4-fluorostyrene) (PSF) as internal standard to circumvent the matrix effects. The three main markers of PS pyrolysis, i.e., styrene, styrene-dimer and styrene-trimer, were monitored. The ratio between the dimer and the trimer significantly varied between the matrices and tended to decrease with the increasing of the Corg in the soil, mainly due to an increased trimer formation. A strong matrix effect affected the slope of the calibration curves by 2 to 8-fold and was correlated with the Corg in the soils. This effect was mitigated when the areas of the markers were normalized by the area of the corresponding marker of PSF. PS of low Mw (Mw 35,000) presented a reduced formation of the three markers compared to PS of high Mw (Mw 400,000), and styrene-dimer was proportionally less formed than the other two markers. Differences in the slopes of calibration curves depended on the marker chosen, highlighting the relevance of selecting the pyrolysis marker in the quantification of microplastics using pyr-GC-MS.