Diversity of synaptic properties is fundamental to brain function, but the molecular basis of this diversity remains elusive. In this study, we developed an immunohistochemical method enabling efficient labeling of synaptic proteins in situ and constructed a super-resolution imaging analysis framework to systematically determine the nanoscale molecular configuration of individual synapses on an unprecedented scale. Our analysis revealed the synapse-type dependent variation in the content and the physical proximity between synaptic proteins, including a presynaptic active zone protein Munc13 and Ca²⁺ channels, in the hippocampus and thalamus. Furthermore, by combining our imaging analysis with optogenetic tools, transgenic mouse lines, and neural activity-dependent labeling techniques, we identified nanoscale molecular changes in synapses associated to long-term synaptic plasticity, circuit development, and memory formation. Our systematic approach to quantify synaptic proteins based on super-resolution imaging will pave the way for understanding causes and significance of synaptic diversity. It will also be particularly useful for identification of molecular targets for treatment of neurological disorders caused by synaptic alternations.