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Luminescence nanothermometry has emerged over the past decade as an exciting field of research due to its potential applications where conventional methods have demonstrated to be ineffective. Preclinical research has been one of the areas that have benefited the most from the innovations proposed in the field. Nevertheless, certain questions concerning the reliability of the technique under in vivoconditions have been continuously overlooked by most of the scientific community. In this proof-of-concept, hyperspectral in vivoimaging is used to explain how unverified assumptions about the thermal dependence of the optical transmittance of biological tissues in the so-called biological windows can lead to erroneous measurements of temperature. Furthermore, the natural steps that should be taken in the future for a reliable in vivoluminescence nanothermometry are discussed together with a perspective view of the field after the findings here reported.