This paper examines the effect of oxidation of asphaltene molecules on the overall polydispersity and rheology of a bituminous matrix: The research hypothesis is that oxidation of asphaltene molecules increases bitumen polydispersity; it is further hypothesized that increased polydispersity leads to reduction in crossover modulus and viscosity of the bituminous matrix. To test this hypothesis, laboratory experiments combined with molecular dynamics simulations were used to examine the properties of bitumen samples containing different concentrations of oxidized asphaltenes. To prepare samples, asphaltene was initially solvent-extracted from the bitumen sample; a portion of the extracted asphaltenes was then oxidized using an ozone treatment oven. The oxidized portion and nonoxidized portion were then blended and added back to the original bitumen sample via solvent blending to ensure uniform distribution. Rheological characterization of bitumen samples containing oxidized asphaltene showed that viscosity measured at shear rates above 1 s(-1) decreases as the concentration of oxidized asphaltenes increases. Our molecular level analysis showed that aggregation of asphaltene molecules increases with the increase in concentration of oxidized asphaltene, which in turn increases the polydispersity in the bitumen matrix. Polydispersity is inversely related to crossover modulus, so an increase in polydispersity is expected to reduce the crossover modulus. The latter was confirmed by our rheometry results showing a significant reduction in crossover modulus when the content of oxidized asphaltene molecules increased. The outcome of this study shows that oxidation of asphaltene molecules alone leads to formation of nanoaggregates of asphaltene that easily move against each other reducing bitumen viscosity at high shear rates. The latter reduction effect may be masked in the field when asphaltene oxidation is accompanied by the loss of volatiles, aromatization, and carbonation as aging progresses. The study highlights the role of oxidized asphaltene molecules in the flow behavior of bitumen; this, in turn, helps refineries and blending terminals tune viscous flow and bitumen rheology by controlling the extent of asphaltene oxidation and subsequent aggregation.