Cryo-electron tomography (cryo-ET) is a powerful technique for imaging biological structures in their native state and in an unperturbed cellular environment. We integrate high resolution imaging by either cryo-ET and sub-tomogram averaging or TYGRESS (Tomography-Guided 3DReconstruction of Subcellular Structures), with comparative genetics, biochemical methods and EM-visible labeling to deconstruct the in situ 3D structure and functional organization of macromolecular complexes. We use cilia and flagella as model systems to advance techniques and approaches for high-resolution imaging of complex cellular structures. Cilia and flagella are conserved and ubiquitous eukaryotic organelles that are composed of more than 600 different proteins and have important biological roles in motility and sensation; defects in their assembly or function cause severe human diseases. The beating of motile cilia and flagella is driven by conformational changes of dynein motors that are coordinated by regulatory complexes. However, the mechanism(s) by which flagellar motility is generated and regulated are largely unknown. Our cryo-ET studies visualize the three-dimensional structures of intact wild-type and mutant flagella, and dissect the organization of key macromolecular complexes. We also study active (beating) flagella that were rapidly frozen to resolve the different conformations of these complexes and identify distinct conformations for different axonemal dyneins and other structures, many of which showing bend direction dependent distributions. Our data provide a molecular blueprint of major ciliary complexes and a new understanding of the distinct roles played by various dyneins and regulatory complexes in the motility of cilia and flagella, suggesting critical modifications to previous hypotheses for the molecular mechanisms underlying the motility of these intriguing nano-machines.