Dystroglycanopathies are a subgroup of muscular dystrophies that arise from defects in the enzymes implicated in the recently elucidated O-mannosylation pathway, resulting in underglycosylation of α-dystroglycan. The emerging identification of additional brain proteins modified by O-mannosylation provides a broader context for interpreting the range of neurological consequences associated with dystroglycanopathies. This form of glycosylation is associated with protein mucin-like domains which present numerous serine and threonine residues as possible sites for modification. Further, the O-Man glycans coexist in this region with O-GalNAc glycans, conventionally associated with such protein sequences, resulting in a complex glycoconjugate landscape. Sorting out the relationships between the various molecular defects in glycosylation and the modes of disease presentation, as well as the regulatory interplay among the O-Man glycans, and the effects on other modes of glycosylation in the same domain is challenging. Here we provide a perspective on chemical biology approaches employing synthetic and analytical methods to address these questions.