Multiphoton microscopy has become a powerful imaging method for minimally invasive evaluation of extracellular matrix (ECM) and cellular structures deep within tissues in their native environments. This technology, which uses ultra-short femto-second laser pulses as the excitation source, is efficient in multiphoton excitation fluorescence (MPEF) of endogenously fluorescent macromolecular systems and induction of highly specific second harmonic generation (SHG) signals from non-centrosymmetric macromolecules such as fibrillar collagens. Both these signals can be captured simultaneously to provide spatially resolved 3D structural organization of ECM as well as cellular morphologies in lung or airway tissue with spectral specificity and sensitivity. These imaging modalities are minimally invasive since structures deep within tissues can be visualized without the need for tissue fixation and/or sectioning. Much of the traditional histological and chemical procedures associated with conventional microscopy methods, which may alter native structure of lung tissue samples, can be circumvented to generate more accurate 3D morphological and fine structural information. In addition to outlining basic principles associated with MPEF and SHG microscopy methods, this review reports potential uses of these high resolution imaging modalities in lung structural imaging. We place special emphasis on imaging 3D structural features of airways, visualizing and quantifying ECM remodeling associated with mouse asthma model as well as the potential uses for multiphoton microscopy in in vitro airway applications.