The Opisthokonta clade includes Metazoa, Fungi, and several unicellular lineages, such as choanoflagellates, ichthyosporeans, and nucleariids. Despite their importance for understanding the origin of animals and fungi, many of these unicellular opisthokonts remain poorly characterized. Here, we report environmental sequences of opisthokonts from diverse aquatic environments and provide a comprehensive phylogenetic analysis of the group.