To realize a thermoelectric power generator, typically, a junction between two materials with different Seebeck coefficients needs to be fabricated. Such differences in Seebeck coefficients can be induced by doping, which renders it difficult when working with two-dimensional (2d) materials. However, doping is not the only way to modulate the Seebeck coefficient of a 2d material. Substrate-altered electron–phonon scattering mechanisms can also be used to this end. Here, we employ the substrate effects to form a thermoelectric junction in ultrathin, few-layer MoS2 films. We investigated the junctions with a combination of scanning photocurrent microscopy and scanning thermal microscopy. This allows us to reveal that thermoelectric junctions form across the substrate-engineered parts. We attribute this to a gating effect induced by interfacial charges in combination with alterations in the electron–phonon scattering mechanisms. This work demonstrates that substrate engineering is a promising strategy for developing future compact thin-film thermoelectric power generators. © 2023, The Author(s).