We attempt to characterize the degree of skin thermal damage by using multiphoton microscopy to characterize dermal thermal damage. Our results show that dermal collagen and elastic fibers display different susceptibility to thermal injury. Morphologically, dermal collagen starts to denature at while fracture and aggregation of elastic fibers do not occur until . With increasing temperatures, the structures of both elastic and collagen fibers deteriorate. While second-harmonic-generation (SHG) imaging is helpful in identifying the denaturation temperature of collagen, autofluorescence (AF) imaging can help to identify the structural alternations of tissue at higher temperatures when SHG signals have decayed. We also employ a ratiometric approach based on the AF-to-SHG index of dermis (ASID) to characterize the degree of dermal thermal damage. Use of the ASID index can bypass the difficulty in analyzing inhomogeneous dermal fibers and show that dermal collagen starts to denature at . Our results suggest that with additional developments, multiphoton microscopy has potential to be developed into an effective in vivo imaging technique to monitor and characterize dermal thermal damage.