Defects have crucial effects on the electronic and optoelectronic properties of two‐dimensional materials. In this work, we measured the changes in the Raman spectra and electrical conductance of monolayer molybdenum disulfide (MoS2) with increasing defects via thermal annealing in a reducing atmosphere. Raman spectra of the basal plane MoS2 on SiO2 differed from those of the edge and revealed intricate changes upon annealing up to 550°C due to the substitution of adsorbed molecules and the creation of highly concentrated defects. On the other hand, MoS2 on hexagonal boron nitride (h‐BN) presented homogenous Raman spectra over the entire area and showed no noticeable changes in its Raman spectra upon annealing. For MoS2 on h‐BN, the charge transfer between MoS2 and adsorbed molecules and the number of defects created by annealing up to 550°C are not sufficient to change the Raman spectra. The distinct behaviors of the two sample spectra are ascribed to different substrate‐induced doping and interfacial adhesion strengths. Thermal annealing effects on the Raman spectra of monolayer MoS2 on SiO2 and h‐BN are investigated. MoS2 on h‐BN shows no noticeable changes in its Raman spectra upon annealing up to 550°C, whereas MoS2 on SiO2 shows intricate changes. The distinct behaviors of the Raman spectra of MoS2 on SiO2 and h‐BN upon annealing are ascribed to different substrate‐induced doping and interfacial adhesion strengths.