Investigating whether landmarks and routes affect navigational efficiency and learning transfer in traffic is essential. In this study, a virtual reality-based driving system was employed to determine the effects of landmarks and routes on human neurocognitive behavior. The participants made four (4) journeys to predetermined destinations. They were provided with different landmarks and routes to aid in reaching their respective destinations. We considered two (2) groups and conducted two (2) sessions per group in this study. Each group had sufficient and insufficient landmarks. We hypothesized that using insufficient landmarks would elicit an increase in psychophysiological activation, such as increased heart rate, eye gaze, and pupil size, which would cause participants to make more errors. Moreover, easy and difficult routes elicited different cognitive workloads. Thus, a high cognitive load would negatively affect the participants when trying to apply the knowledge acquired at the beginning of the exercise. In addition, the navigational efficiency of routes with sufficient landmarks was remarkably higher than that of routes with insufficient landmarks. We evaluated the effects of landmarks and routes by assessing the recorded information of the drivers’ pupil size, heart rate, and driving performance data. An analytical strategy, several machine learning algorithms, and data fusion methods have been employed to measure the neurocognitive load of each participant for user classification. The results showed that insufficient landmarks and difficult routes increased pupil size and heart rate, which caused the participants to make more errors. The results also indicated that easy routes with sufficient landmarks were deemed more efficient for navigation, where users’ cognitive loads were much lower than those with insufficient landmarks and difficult routes. The high cognitive workload hindered the participants when trying to apply the knowledge acquired at the beginning of the exercise. Meanwhile, the data fusion method achieved higher accuracy than the other classification methods. The results of this study will help improve the use of landmarks and design of driving routes, as well as paving the way to analyze traffic safety using the drivers’ cognition and performance data.