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 laver. Segregation occurs for S up to 8 nm at 510 ° C, this value being reduced by ∼ 50% at 450 ° C. © 2005 Elsevier B.V. All rights reserved.