Cancers rank among the most life-threatening conditions for humans. Resection surgery remains one of the most prevalent treatments. Intraoperative tumor margin assessment plays a crucial role in the success of surgical resection, influencing recurrence rates and patient prognosis. This importance is exemplified for brain tumors due to the complexities of the anatomical and functional boundaries. Given the inherent limitations of traditional intraoperative guidance techniques, marked by suboptimal imaging resolution, shallow tissue penetration depths and sluggish imaging speeds, there is an urgent imperative for novel and effective imaging methodologies to enhance both accuracy and expediency to shorten surgical time and reduce inadvertent omission of imperceptible tumor cells. Among the emerging guidance techniques, photoacoustic imaging (PAI) stands out for its potential to provide high-speed, high-resolution 3D imaging at large tissue depths. PAI’s ability to visualize molecular and structural changes, such as hypoxia, lipid variation, and nuclear morphology, makes it a compelling tool for differentiating malignant tissues from normal tissues during surgery. This chapter reviews recent advances in PAI for intraoperative tumor margin assessment, focusing imaging configurations such as clinical array-based PAI systems, ultraviolet photoacoustic microscopy (PAM), and handheld PAM. It also explores the integration of PAI with complementary imaging techniques, such as ultrasound and optical imaging modalities, to enhance comprehensive tumor assessment. Finally, this chapter highlights the potential of PAI to transform surgical oncology by reducing re-excision rates, improving patient outcomes, and shaping the future of intraoperative imaging.