This study aimed to explore the relationship between eye movements and cognitive functions when recognizing destinations in guide signs using actual on-board videos and the effects of driving speed and location of guide signs on eye movements. Twenty-four participants were recruited. Attention and processing speed, verbal memory, visual memory, planning, and useful field of view (UFOV) were assessed as cognitive functions necessary for driving. Eye movements while recognizing the destination in guide signs (duration and number of fixations and amplitude and number of saccades) were measured using four conditions in on-board videos: slow/fast speed, front/left side. The results showed that cognitive functions correlated during in-front conditions at slow and fast speeds. At left-side guide signs, cognitive functions did not correlate, but correlated with age and driving experience. The results suggest that recognizing left-side guide signs required a higher skill, which depended more on the driver’s experience than on cognitive functions. In addition, of the four conditions, visual recognition of destinations in guide signs made more difficult with fast speeds and left-side locations. The increased load from left-side guide signs and faster speeds influenced eye movements. Thus, the location of the guide sign and the speed was shown to affect the difficulty in recognizing destinations within the guide signs.