This study aims to explore the influential sources of organic matter in first flush runoff from urban roadways by comparing organic carbon content and particle size distribution in road dust with those from discharge from vehicles during rainfall. Samples on first flush runoff and road dust were collected from urban roadways. In addition, vehicle drainage was assumed to flow from vehicles during rainfall events, so vehicle wash-off water was collected by spraying water onto the top and from the underside of vehicles to simulate accumulation during a vehicle run. In road dust, the organic carbon content in the <0.2 mm fraction was about twice that of the 0.2–2 mm fraction. The particle size distributions of both first flush runoff and vehicle wash-off water were similar, and particles <0.2 mm contributed to over 95% of the total volume. The dissolved organic carbon concentration in the vehicle wash-off water was considerably higher than that in the road dust/water mixture. The total organic carbon content in road dust was positively correlated with annual daily traffic. Therefore, vehicles were thought to strongly influence the nature of road dust.