Post-stroke navigation complaints are frequent (about 30%) and intervention is possible, but there is no assessment instrument to identify patients with navigation complaints. We therefore studied the clinical validity of the Wayfinding Questionnaire (WQ) in a cross-sectional study with 158 chronic stroke patients and 131 healthy controls. Patients with low (more navigation complaints) versus normal WQ scores were compared for demographics, stroke characteristics, emotional and cognitive complaints, and health-related quality of life (HRQoL). Actual navigation performance of 78 patients was assessed in a virtual reality setting. Effect sizes (d) were calculated. WQ responses (22 items) of stroke patients were compared with those of controls (discriminant validity). Results showed that patients with a low WQ score (n = 49, 32%) were more often women (p = 0.013) and less educated (p = 0.004), reported more cognitive complaints (d = 0.69), more emotional problems (d = 0.38 and 0.52), and lower HRQoL (d = 0.40 and 0.45) and, last but not least, performed worse on the navigation ability tasks (d = 0.23-0.80). Patients scored lower than controls on 21/22 WQ items, predominantly with small to medium effect sizes (d = 0.20-0.51). We conclude that the WQ is valid as a measure of navigation complaints in stroke patients, and thus strongly advocate its use in stroke care.