A method of manufacturing a thin film transistor array panel is provided, which includes: forming a semiconductor layer of polysilicon on an insulating substrate; forming a gate insulating layer on the semiconductor layer; forming a gate electrode on the gate insulating layer; forming a source region and a drain region by doping conductive impurities in the semiconductor layer; forming an interlayer insulating layer covering the gate electrode; forming a source electrode and a drain electrode respectively connected to the source and the drain regions; forming a passivation layer covering the source and the drain electrodes; forming a pixel electrode connected to the drain electrode; and forming a first alignment key when forming one selected from the semiconductor layer, the gate electrode, the source and the drain electrodes, and the pixel electrode, wherein one selected from the semiconductor layer, the gate electrode, the source and the drain electrodes, and the pixel electrode is at least formed by photolithography process using a photoresist pattern as an etch mask, and a second alignment key completely covering the first alignment key is formed at the same layer as the photoresist pattern.