The penta‐subunit retromer complex of yeast mediates selective retrieval of membrane proteins from the prevacuolar endosome to the trans Golgi network. In this study, we set out to generate a panel of vps35 dominant‐negative mutants that disrupt retromer‐mediated cargo sorting. Mapping of the mutations revealed two types of alterations leading to dominant‐negative behavior of the 944‐amino acid protein: (i) mutations at or near the R98 residue or (ii) C‐terminal truncations exemplified by a nonsense mutation at codon 733. Both could be suppressed by overexpression of wild‐type Vps35p, suggesting that these dominant‐negative mutants compete for interactions with other retromer subunits. Interestingly, Vps35‐R98W expression destabilized Vps26p while having no effect on Vps29p stability, while Vps35‐Q733* expression affected Vps29p stability but had no effect on Vps26p. Measurement of Vps35/Vps26 and Vps35/Vps29 pairwise associations by coimmunoprecipitation in the presence or absence of other retromer subunits indicated that the R98 residue, which is part of a conserved PRLYL motif, is critical for Vps35p binding to Vps26p, while both R98 and residues 733–944 are needed for efficient binding to Vps29p.