The mammary gland is thought to have evolved from the apocrine sweat gland and has undergone significant evolutionary transformations. However, the evolution of the mammary gland is still underexplored due to limited model systems. Here, Gat Rauner, Charlotte Kuperwasser and colleagues generate 3D organoids from a range of species to explore the evolution and function of the mammary gland. First, the authors grow mammary gland organoids from eight eutherian mammals and the opossum, which is part of the marsupial group that diverged from eutherians about 166 million years ago. Cells isolated from adult mammary glands of these species form branched epithelial structures and ductal-lobular networks in a 3D matrix. Then, the authors explore what factors are necessary for the generation of the organoids. They find that Rho-associated, coiled-coil-containing protein kinase (ROCK) is involved in regulating organoid branching. Adding or removing ROCK inhibitor from the media affects organoid branching differently in different species. Overall, this study establishes an experimental platform for culturing 3D mammary gland organoids from a range of species and demonstrates the utility of 3D organoid models in understanding the evolutionary processes in mammalian development.