AbstractMolecular phylogenomics investigates evolutionary relationships based on genomic data. However, despite genomic sequence conservation, changes in protein interactions can occur relatively rapidly and may cause strong functional diversification. To investigate such functional evolution, we here combine phylogenomics with interaction proteomics. We develop this concept by investigating the molecular evolution of the shelterin complex, which protects telomeres, across 16 vertebrate species from zebrafish to humans covering 450 million years of evolution. Our phylointeractomics screen discovers previously unknown telomere-associated proteins and reveals how homologous proteins undergo functional evolution. For instance, we show that TERF1 evolved as a telomere-binding protein in the common stem lineage of marsupial and placental mammals. Phylointeractomics is a versatile and scalable approach to investigate evolutionary changes in protein function and thus can provide experimental evidence for phylogenomic relationships.