Protein tyrosine kinases (PTKs) are implicated in cell proliferation, differentiation, and receptor-mediated signalling events. Recruitment of intracellular PTKs into the signalling complex, often localized at the inner surface of the cell membrane, involves SH2 and SH3 domains attached to the catalytic kinase domain. While the interaction of SH2 and SH3 domains with their target sequences is well documented in a number of cases, the contribution of the catalytic domain itself in conferring specificity to a given signal cascade is not fully understood. We addressed this question and employed the phage display technique to assess the substrate requirements for the highly related Src-like PTKs c-Src, Blk, Lyn and the distantly related Syk. A diverse peptide library on phage was established, and after multiple rounds of phosphorylation and selection of phage displaying phosphotyrosine-containing peptides, canonical substrate sequences for each of the PTKs were enriched. The PTKs Blk and Lyn implicated in B cell signalling were found to prefer peptide substrates of the structure I/L-Y-D/E-X-L which resemble critical features of the ITAM motifs found in, e.g. the intracellular components Ig-alpha and Ig-beta of the beta cell receptor. All Src-like PTKs had a requirement for isoleucine or leucine in the position -1 with respect to the phosphorylated tyrosine residue in position 0. While Blk and Lyn had a strong preference for a negatively charged amino acid in position +1, c-Src preferred tryptophan or glycine in this position. Syk, not belonging to the Src-like PTK family, revealed a distinct substrate requirement for aspartic acid in position -1 and glutamic acid in position +1. In general, all PTKs we have tested had a strong preference for a particular amino acid in the positions -1 and +1 adjacent to the tyrosine residue.