Thermoacoustic imaging (TAI) is an innovative imaging modality that combines microwave and ultrasound technologies. TAI utilizes the thermoelastic effect to convert microwave energy into acoustic signals, thereby enabling high-contrast imaging of biological tissues with deep penetration. In the electromagnetic spectrum, microwaves (ranging from hundreds of MHz to several GHz) have wavelengths on the order of centimeters to millimeters, which significantly reduces their interaction with cellular and subcellular structures. Hence, they primarily interact with tissue on a macroscopic scale. Tumors, blood clots, and foreign bodies have dielectric properties that differ from healthy tissue and non-coagulated blood, leading to high contrast imaging. TAI’s ability to achieve deep tissue penetration and exceptional contrast makes it particularly effective for many medical and biomedical applications. In preclinical research, TAI has shown significant potential for visualizing anatomical structures and tracking disease progression. Applications include breast, prostate, and brain cancer detection and characterization, brain hemorrhage analysis, and joint imaging. TAI has also been utilized for detection of foreign bodies. Despite its advantages, TAI faces technical challenges toward wider implementation, including detection sensitivity optimization, and hardware complexity. For example, there are currently no TAI systems for preclinical research that are commercially available. This chapter explores the fundamental principles of TAI, its core system components, and its diverse preclinical applications. By addressing current challenges and highlighting advancements, this work underscores TAI’s potential in biomedical research and diagnostics. Future directions include integrating TAI with machine learning and multimodal imaging systems to enhance its clinical and research utility.