LAMMER protein kinases are ubiquitous throughout eukaryotes, including multiple paralogues in mammals. Members are characterized by similar overall structure and highly identical amino acid sequence motifs in catalytic subdomains essential for phosphotransfer and interaction with substrates. LAMMER kinases phosphorylate and regulate the activity of the SR protein class of pre-mRNA splicing components, both in vitro and in vivo. In this study, we define an optimum in vitro consensus phosphorylation site for three family members using an oriented degenerate peptide library approach. We also examine the substrate specificity and interactions of several LAMMER protein kinases from widely diverged species with potential substrates, including their own N-termini, predicted to be substrates by the peptide-based approach. Although the optimal in vitro consensus phosphorylation site for these kinases is remarkably similar for short peptides, distinct substrate preferences are revealed by in vitro phosphorylation of intact proteins. This finding suggests that these kinases may possess varied substrates in vivo, and thus the multiple LAMMER kinases present in higher eukaryotes may perform differentiable functions. These results further demonstrate that these kinases can phosphorylate a number of substrates in addition to SR proteins, suggesting that they may regulate multiple cellular processes, in addition to the alternative splicing of pre-mRNAs.