This paper estimates the impact of road tolls in the road freight transportation cost. To this effect, the authors conducted a multifactorial regression analysis to study the effect of distance¿considering Freight Trip Length Distribution¿and tolls between origin-destination pairs on the trucking operating costs. To do so, the authors used information from the Department of Transportation of Colombia¿s information system to estimate efficient costs for trucks (SICE-TAC). This system analyzes the operational costs for small, medium and large trucks, considering fixed, variable, and additional costs, including road tolls. The results indicate that the toll cost of a route could be explained by the total distance between the origin-destination pair for the different truck types, where the longer the distance, the higher the toll cost. The analyses also indicate that in average the toll cost ranges between 5% (small trucks) to 9% (medium-large trucks) of the freight operational costs