G-protein-coupled receptors (GPCRs) are key cellular signaling proteins and have been targeted by approximately 30–40% of marketed drugs for treating many human diseases including cancer and heart failure. Recently, we directly observed activation of the M2 muscarinic receptor through long-timescale accelerated molecular dynamics (aMD) simulation, which revealed distinct inactive, intermediate and active conformers of the receptor. Here, FTMAP is applied to search for ‘hot spots’ in these activation-associated conformers using a library of 16 organic probe molecules that represent fragments of potential drugs. Seven allosteric (non-orthosteric) binding sites are identified in the M2 receptor through the FTMAP analysis. These sites are distributed in the solvent-exposed extracellular and intracellular mouth regions, as well as the lipid-exposed pockets formed by the transmembrane α helices TM3-TM4, TM5-TM6 and TM7-TM1/TM2. They serve as promising target sites for designing novel allosteric modulators as receptor-selective drugs.