A display substrate and a manufacturing method thereof, and a display device are provided. The display substrate includes a base substrate, and a shift register unit, a first clock signal line, and a second clock signal line in a peripheral region; a first portion and a second portion of a first connection wire of the display substrate are connected to a first gate electrode of a first control transistor of the shift register unit; an orthographic projection of the first portion on the base substrate is on a side of an orthographic projection of an active layer of the first control transistor on the base substrate away from a display region; and an orthographic projection of the second portion on the base substrate is on a side of the orthographic projection of the active layer of the first control transistor on the base substrate close to the display region.