Eukaryotic cells develop asymmetric shapes suited for specific physiological functions. Morphogenesis of polarized domains and structures requires the amplification of molecular asymmetries by scaffold proteins and regulatory feedback loops. Small monomeric GTPases signal polarity, but how their downstream effectors and targets are spatially co-ordinated to break cell symmetry is poorly understood. Septins comprise a novel family of GTPases that polymerize into non-polar filamentous structures which scaffold and restrict protein localization. Recent studies show that septins demarcate distinct plasma membrane domains and cytoskeletal tracks, enabling the formation of intracellular asymmetries. Here, we review these findings and discuss emerging mechanisms by which septins promote cell asymmetry in fungi and animals.