Indium segregation during the growth of multilayer InAs/GaAs(001) quantum dot (QD) structures has been studied using reflection high-energy electron diffraction (RHEED) measurements of the critical coverage (θ crit ) for second layer QD formation. A model bilayer structure was used in order to separate the effects of segregation and strain. The structure comprises an upper QD layer formed on top of a buried two-dimensional InAs layer. Growth temperature and the GaAs spacer layer thickness (S) are both found to have a significant effect on θ crit . Indium segregation during growth of the capping layer leads to the presence of a surface In adatom population prior to deposition of the second InAs layer. Segregation occurs for S up to 8nm at 510°C, this value being reduced by ∼50% at 450°C.