Tumor hypoxia is an important factor in assessment of both cancer progression and cancer treatment efficacy. This has driven a substantial effort toward development of imaging modalities that can directly measure oxygen distribution and therefore hypoxia in tissue. Although several approaches to measure hypoxia exist, direct measurement of tissue oxygen through an imaging approach is still an unmet need. To address this, we present a new approach based on in vivo application of photoacoustic lifetime imaging (PALI) to map the distribution of oxygen partial pressure (pO2 ) in tissue. This method utilizes methylene blue, a dye widely used in clinical applications, as an oxygen-sensitive imaging agent. PALI measurement of oxygen relies upon pO2 -dependent excitation lifetime of the dye. A multimodal imaging system was designed and built to achieve ultrasound (US), photoacoustic, and PALI imaging within the same system. Nude mice bearing LNCaP xenograft hindlimb tumors were used as the target tissue. Hypoxic regions were identified within the tumor in a combined US/PALI image. Finally, the statistical distributions of pO2 in tumor, normal, and control tissues were compared with measurements by a needle-mounted oxygen probe. A statistically significant drop in mean pO2 was consistently detected by both methods in tumors.