Values stored in the route key item for a route feature class may or may not be unique. For routes stored in personal or ArcSDE geodatabases, locating route events will be faster if the route key values are unique. This setting has no effect for routes stored in coverages and shapefiles.
If the data does not have unique route key values or you are unsure, you should not check this option as the segmentation algorithm will only look for one route with the corresponding route key. The event being processed may or may not locate itself on the route feature that was found.