The examined samples were W-Re and Mo-Re thin alloy layers with high content of Rhenium, applied in industry as protective coatings. The 550 nm of W-50.4%Re and 580 nm of Mo-39.5%Re alloys were deposited on crystalline silicon and amorphous silica substrates by magnetron sputtering method. For thermal characterization of investigated layers the scanning thermal microscopy (SThM) and high frequency photothermal radiometry (HF-PTR) measurements were carried out. The thermal methods were supported by the microstructural studies performed by the atomic force microscopy (AFM) and X-ray diffraction (XRD). The SThM method allowed determining the thermal conductivity (κ) of the alloy layers. From the HF-PTR the effective thermal conductivity (κeff) was determined directly. Estimation of the thermal boundary resistance (Rth) between particular alloy layer and its substrate enabled determination of the real κ of the layer with its thermal diffusivity (α) and effusivity (ε) from the HF-PTR fitting. The results showed, that W-Re and Mo-Re layers deposited on Si are characterized by higher κ, α, and ε values, and lower Rth in comparison to those deposited on SiO2. The κ values for W-Re and Mo-Re layers deposited on Si and SiO2 substrates were estimated in the range from 7 W·m−1 K−1 to 12 W·m−1 K−1, and 1.6 W·m−1 K−1 to 4 W·m−1 K−1, respectively. The correlation between the alloy layer structure, it's thermophysical properties and substrate structure was observed. The AFM and XRD measurements confirmed the short range order formation in Mo-Re layers deposited on Si.