A positioning substrate is used for performing a teaching operation on a transfer mechanism for transferring a target substrate in a semiconductor processing system. The positioning substrate includes a substrate body made of a material selected from the group consisting of the same material as the target substrate, semiconductor, compound semiconductor, and ceramic. The substrate body has an outer contour sized to be handled by the transfer mechanism as an alternative to the target substrate. The positioning substrate also includes a positioning assistant having a combination of a positioning hole and a positioning reference line formed in the substrate body. The positioning hole is formed to penetrate the substrate body in a thickness direction. The positioning reference line is formed on a surface of the substrate body to extend across an opening of the positioning hole and have a predetermined width.