Ubiquitin (Ub) is a small protein modifier that regulates many biological processes, including gene transcription, cell-cycle progression, DNA repair, apoptosis, virus budding and receptor endocytosis. Ub can be conjugated to target proteins either as a monomer or as Ub chains that vary in length and linkage type. The various types of Ub modification are linked to distinct physiological functions in cells. MonoUb, for example, regulates DNA repair and receptor endocytosis, whereas lysine 48-linked Ub chains label proteins for proteasomal degradation. More recently, the importance of chains conjugated through the other six lysines in Ub, known as atypical Ub chains, has been revealed. Atypical chains can be homotypic, sequentially using the same lysine residue in Ub for conjugation; mixed-linkage, utilizing several distinct lysines to connect consecutive Ub moieties; or heterologous, connecting Ub with other Ub-like modifiers. Here, we describe recent progress in the understanding of atypical Ub chain assembly and their recognition by Ub-binding domains, and we discuss further their functional roles in vivo.