This paper presents various methods of estimating the full marginal cost (FMC) of highway passenger transportation. First, the computation of FMC is performed using the marginal cost functions, most of which were developed by Ozbay et al. [Ozbay, K., Bartin, B., Berechman, J., 2001. Estimation and evaluation of full marginal costs of highway transportation in New Jersey. Journal of Transportation and Statistics 4 (1)]. FMC is defined and calculated as “total cost per trip” as explained in this paper. However, in multiple origin-destination and multiple route highway networks, the practical application of the network-wide FMC concept is complicated. These issues are addressed in detail in this paper. Therefore, in the second method, a multiple route based FMC approach is proposed for a given origin-destination pair in the network. It is observed that the marginal values of different paths vary as much as 28%. Third, a comparison of FMC estimation results of two distinct measurement tools is presented. The FMC estimation is performed between a selected OD pair using the static transportation planning software output (TransCAD). The same analysis is repeated using the stochastic traffic simulation software output (PARAMICS). The differences in FMC values estimated by static transportation planning software and microscopic traffic simulation software are discussed.