<p>Recurrent transitions in feeding strategies have shaped the eukaryotic tree of life, as unrelated groups independently evolved similar solutions to common ecological challenges. One of the most interesting yet poorly studied of these shifts is the transition towards osmotrophy. We reconstructed the evolution of four eukaryotic groups that specialized in osmotrophy through convergent evolution. Here we show that these groups arose most likely during the Tonian period (1,000–720 million years ago) or slightly before, and possess a genetic toolkit enriched in shared metabolic functions. We report signatures that are compatible with horizontal gene transfer encompassing at least 20% of this toolkit. Phylogenetic reconciliation analyses show that this fraction of the toolkit ranks in the upper percentiles for inferred horizontal gene transfers, particularly in the period in which the osmotrophic groups originated. Moreover, analyses of the total gene content using supervised phylogenetic screening identified 166 gene tree topologies that are supportive of transfer events involving distantly related eukaryotic osmotrophs. These data include transfer highways between Fungi and Pseudofungi and between Labyrinthulea and Teretosporea. Our work thus unravels the evolutionary history of four independent transitions towards specialization in osmotrophy within the eukaryotes, supporting a role of gene transfer in the evolution of these groups.</p>