Due to the dramatic difference in spatial resolution between the central fovea and the surrounding retinal regions, accurate fixation on important objects is critical for human visual behavior. It is known that the preferred retinal location for fixation (PRL) of healthy human observers does not exactly coincide with the retinal location with the highest cone density. It is not currently known, however, whether the PRL is consistent within an observer or subject to fluctuations and, moreover, whether observers’ subjective fixation location coincides with the PRL. We studied whether the PRL changes between days. We used an adaptive optics scanning laser ophthalmoscope to project a Maltese cross fixation target on an observer’s retina, and continuously imaged the exact retinal location of the target, while the observers fixated on it. We found that observers consistently use the same PRL across days, regardless of how much the PRL is displaced from the cone density peak location. We then showed observers small stimuli near the visual field location they were asked to fixate on, and the observers judged whether the stimuli appeared along their line of sight (i.e., in fixation) or not. Observers’ precision in this task approached that of fixation itself. Observers based their judgement on both the external world coordinates and the retinal location of the stimuli. We conclude that, for a monocular fixation task, the PRL in a normally functioning visual system is fixed, and observers are relatively well aware of its location.