Mapping tumor heterogeneity and hypoxia within a living intact organism is essential for understanding the processes involved in cancer progression and assessing long-term responses to therapies. Efficient investigations into tumor hypoxia mechanisms have been hindered by the lack of intravital imaging tools capable of multiparametric probing of entire solid tumors with high spatial and temporal resolution. Here, we exploit volumetric multispectral optoacoustic tomography (vMSOT) for accurate, label-free delineation of tumor heterogeneity and dynamic oxygenation behavior. Mice bearing orthotopic MDA-MB-231 breast cancer xenografts were imaged noninvasively during rest and oxygen stress challenge, attaining time-lapse three-dimensional oxygenation maps across entire tumors with 100 mm spatial resolution. Volumetric quantification of the hypoxic fraction rendered values of 3.9% to 21.2%, whereas the oxygen saturation (sO(2)) rate declined at 1.7% to 2.3% permmin all tumors when approaching their core. Three distinct functional areas (the rim, hypoxic, and normoxic cores) were clearly discernible based on spatial sO(2) profiles and responses to oxygen challenge. Notably, although sO(2) readings were responsive to the challenge, deoxyhemoglobin (HbR) trends exhibited little to no variations in all mice. Dynamic analysis further revealed the presence of cyclic hypoxia patterns with a 21% average discrepancy between cyclic fractions assessed via sO(2) (42.2% +/- 17.3%) and HbR fluctuations (63% +/- 14.1%) within the hypoxic core. These findings corroborate the strong potential of vMSOT for advancing preclinical imaging of cancer and informing clinical decisions on therapeutic interventions.Significance: vMSOT provides quantitative measures of volumetric hypoxic fraction and cyclic hypoxia in a label-free and noninvasive manner, providing new readouts to aid tumor staging and treatment decision making.