We perform in-situ two-cycle thermal cycling and annealing studies for a transferred CVDgrown monolayer MoS2 on a SiO2/Si substrate, using spatially resolved micro-Raman and PL spectroscopy. After the thermal cycling and being annealed at 305 °C twice, the film morphology and film-substrate bonding are significantly modified, which together with the removal of polymer residues cause major changes in the strain and doping distribution over the film, and thus the optical properties. Before annealing, the strain associated with ripples in the transferred film dominates the spatial distributions of the PL peak position and intensity over the film; after annealing, the variation in film-substrate bonding, affecting both strain and doping, becomes the leading factor. This work reveals that the film-substrate bonding, and thus the strain and doping, is unstable under thermal stress, which is important for understanding the substrate effects on the optical and transport properties of the 2D material and their impact on device applications.