Hyperthermia is a cancer treatment modality that could be delivered as a stand-alone treatment or in conjunction with chemotherapy or radiation therapy. Noninvasive and real-time temperature monitoring of the heated tissue improves the efficacy and safety of the treatment. Ultrasound-based thermometry requires a temperature-sensitive acoustic parameter that can be used to estimate the temperature by tracking changes in that parameter during heating. This dissertation describes the experiments and simulations performed to obtain the temperature dependence of acoustic harmonics generated by nonlinear ultrasound propagation in several media including: water, an attenuating tissue-mimicking liquid, ex vivo bovine muscle tissues, and tissue-mimicking gel phantoms. The mechanisms of action of harmonic generation in water and in the attenuating liquid, made by a mixture of 90% glycerol and 10% water (by volume), as a function of temperature at various frequencies have been investigated using a temperature dependent Khokhlov–Zabolotskaya–Kuznetsov (KZK) nonlinear acoustic wave propagation model. The simulation results were compared with and validated by measurements. In water, the harmonic amplitudes decrease with increasing the temperature at low frequencies (1 and 3.3 MHz), while the opposite temperature dependence was observed at higher frequencies (13 and 20 MHz). The harmonic generation significantly increased with temperature in the tissue-mimicking liquid at both frequencies of 5 and 13 MHz. The temperature dependence of harmonics in tissue-mimicking gel phantoms and ex vivo bovine muscle tissues were measured using a commercial high-frequency ultrasound imaging system, and a new noninvasive ultrasound-based thermometry has been developed that is based on the backscattered energy of the harmonics. The sensitivity of this new thermometry technique to medium’s motion was studied and compared with the conventional echo-shift thermometry technique. Based on this study, it is suggested that noninvasive temperature estimation is feasible using acoustic harmonics with lower sensitivity to motion artifacts compared to the conventional echo-shift technique.