The hindered stepwise aggregation (HSA) model was used to elucidate the molecular aggregation in heavy petroleum fractions which were derived from supercritical fluid extraction fractionation (SFEF) of Venezuela Orinoco vacuum residue (VR). The SFEF fractions consisted of multiple extractable narrow fractions and a nonextractable end-cut. The SFEF fractions were diluted with toluene, and their number-average molecular weights (MWs) were determined using vapor pressure osmometry (VPO). The initial molecular association constants (K-1) and aggregation hindrance factors (H) of the HSA model for each SFEF fraction were calculated from the VPO MWs at various SFEF solution concentrations. The results showed that the HSA model fit well with VPO MW data and the parameters of the HSA model are physically significant. The values of MW and K-1 increased as the SFEF fraction became heavier. The SFEF end-cut had the highest K-1 and lowest H value, in which the aggregates were 2 to 8 monomers. Except for the initial fraction, all the SFEF fractions formed aggregates at solution concentrations higher than 30 g/L. The value of K-1 was dependent on the number of aromatic rings, whereas H is dependent on the size of aromatic ring and side-chain length. The VPO MWs of light SFEF fractions were in agreement with those determined from electrospray ionization (ESI) mass spectrometry (MS) or gel permission chromatography (GPC). The VPO MWs of the highly aggregated SFEF fractions were higher than those of ESI MS due to low ionization efficiency but were much lower than those of GPC.