The dystroglycan gene produces two products from a single mRNA, the extracellular α-dystroglycan and the transmembrane β-dystroglycan. The Duchenne muscular dystrophy protein, dystrophin, associates with the muscle membrane via β-dystroglycan, the WW domain of dystrophin interacting with a PPxY motif in β-dystroglycan. A panel of four monoclonal antibodies (MANDAG1–4) was produced using the last 16 amino acids of β-dystroglycan as immunogen. The mAbs recognized a 43 kDa band on Western blots of all cells and tissues tested and stained the sarcolemma in immunohistochemistry of skeletal muscle over a wide range of animal species. A monoclonal antibody (mAb) against the WW domain of dystrophin, MANHINGE4A, produced using a 16-mer synthetic peptide, recognized dystrophin on Western blots and also stained the sarcolemma. We have identified the precise sequences recognized by the mAbs using a phage-displayed random 15-mer peptide library. A 7-amino-acid consensus sequence SPPPYVP involved in binding all four β-dystroglycan mAbs was identified by sequencing 17 different peptides selected from the library. PPY were the most important residues for three mAbs, but PxxVP were essential residues for a fourth mAb, MANDAG2. By sequencing five different random peptides from the library, the epitope on dystrophin recognized by mAb MANHINGE4A was identified as PWxRA in the first β-strand of the WW domain, with the W and R residues invariably present. A recent three-dimensional structure confirms that the two epitopes are adjacent in the dystrophin–dystroglycan complex, highlighting the question of how the two interacting motifs can also be accessible to antibodies during immunolocalization in situ.