A display substrate includes a base substrate, a switching device on the base substrate and an alignment pattern. The switching device includes an active pattern, a gate insulation layer pattern partially covering the active pattern, a gate electrode on the gate insulation layer pattern, and a source electrode and a drain electrode electrically connected to the active pattern. The alignment pattern has a multi-layered structure and is spaced apart from the switching device on the base substrate. The alignment pattern includes materials which have different transmittances.