Neural circuit function depends on the pattern of synaptic connections between neurons and the strength of those connections. Synaptic strength is determined by both postsynaptic sensitivity to neurotransmitter and the presynaptic probability of action potential evoked transmitter release (P_r). Whereas morphology and neurotransmitter receptor number indicate postsynaptic sensitivity, presynaptic indicators and the mechanism that sets P_r remain to be defined. To address this, we developed QuaSOR, a super-resolution method for determining P_r from quantal synaptic transmission imaging at hundreds of glutamatergic synapses at a time. We mapped the P_r onto super-resolution 3D molecular reconstructions of the presynaptic active zones (AZs) of the same synapses at the Drosophila larval neuromuscular junction (NMJ). We find that P_r varies greatly between synapses made by a single axon, quantify the contribution of key AZ proteins to P_r diversity and find that one of these, Complexin, suppresses spontaneous and evoked transmission differentially, thereby generating a spatial and quantitative mismatch between release modes. Transmission is thus regulated by the balance and nanoscale distribution of release-enhancing and suppressing presynaptic proteins to generate high signal-to-noise evoked transmission.