An orthotopic mouse mammary window chamber (MWC) model has been developed for multimodal in-vivo functional and anatomical imaging of breast cancer xenografts. Capabilities to image numerous physiological aspects of the same tumor microenvironment over time has important applications such as in experiments studying the efficacies of therapeutic interventions, improvement of cancer detection and investigating basic cancer biology. The compatibility of this MWC model with optical, nuclear and magnetic resonance imaging (MRI) makes it possible to perform a multitude of studies ranging from cellular imaging to whole body imaging. Thus, the MWC represents a powerful tool for breast cancer research. Here, two imaging applications are highlighted, namely the nuclear imaging of glycolytic metabolism with 18FFDG and MRI of tissue perfusion. Nuclear imaging is performed with the use of a 3μm thin phosphor scintillator placed directly in contact with the tissue and visible light from the scintillation is directly detected in a low noise, light tight imaging system. Tissue perfusion is imaged either qualitatively with a dynamic contrast enhancement (DCE) MRI technique or quantitatively with an arterial spin labeling flow-sensitive alternating inversion recovery-rapid acquisition with relaxation enhancement (FAIR-RARE) technique.