•Cryo-electron tomography (cryoET) subtomogram averaging has emerged as a structural biology method for sparse and heterogenerous sampls.•CryoET subtomogram averaging enables in situ structure determination.•CryoET subtomogram classification can delineate different conformational states of macromolecular complexes.•Future developments in cryoET and correlative super resolution microscopy promises to bring unprecedented integration of cell biology and structural biology. Cryo-electron tomography (cryoET) can provide 3D reconstructions, or tomograms, of pleomorphic objects such as organelles or cells in their close-to-native states. Subtomograms that contain repetitive structures can be further extracted and subjected to averaging and classification to improve resolution, and this process has become an emerging structural biology method referred to as cryoET subtomogram averaging and classification (cryoSTAC). Recent technical advances in cryoSTAC have had a profound impact on many fields in biology. Here, I review recent exciting work on several macromolecular assemblies demonstrating the power of cryoSTAC for in situ structure analysis and discuss challenges and future directions.