A self-assembled monolayer (SAM) consisting of a mixture of CH3S–Au–SCH3, CH3S–Au–S(CH2)2CH3, and CH3(CH2)2S–Au–S(CH2)2CH3 was studied systematically using scanning tunneling microscopy and density functional calculations. We find that the SAM is subjected to frequent changes at the molecular level on the time scale of ∼minutes. The presence of CH3S or CH3S–Au as a dissociation product of CH3S–Au–SCH3 plays a key role in the dynamical behavior of the mixed SAM. Slow phase separation takes place at room temperature over hours to days, leading to the formation of methyl-thiolate-rich and propyl-thiolate-rich phases. Our results provide new insights into the chemistry of the thiolate–Au interface, especially for ligand exchange reaction in the RS–Au–SR staple motif.