In vivo imaging is an important tool for pre-clinical studies of lung function and disease. The widespread availability of multimodal animal imaging systems and the rapid rate of diagnostic contrast agent development has empowered researchers to non-invasively study lung function and pulmonary disorders. Investigators can identify, track, and quantify biological processes over time. In this review, we highlight the fundamental principles of bioluminescence, fluorescence, planar X-ray, X-ray computed tomography (CT), magnetic resonance imaging (MRI), and nuclear imaging modalities (such as positron emission tomography and single photon emission computed tomography; PET and SPECT) that have been successfully employed for the study of lung function and pulmonary disorders in a pre-clinical setting. The major principles, benefits, and applications of each imaging modality and technology are reviewed. Limitations and the future prospective of multimodal imaging in pulmonary physiology are also discussed. In vivo imaging bridges molecular biological studies, drug design and discovery, and the imaging field with modern medical practice, and as such, will continue to be a mainstay in biomedical research.