Sex chromosomes in males of most mammalian species share only a small homologous segment, the pseudoautosomal region (PAR), wherein double-strand break (DSB) formation must occur for correct meiotic segregation. We find that multiple DSB-promoting factors hyperaccumulate in the PAR, including the ankyrin-repeat domain-containing protein ANKRD31. We used immunoprecipitation and mass spectrometry from extracts of juvenile mouse testes to identify potential ANKRD31-interacting proteins.