The NSD1 protein lysine methyltransferase (PKMT) was known to methylate H3K36. We show here that NSD1 prefers aromatic, hydrophobic and basic residues at the -2, -1 and +2, and +1 sites of its substrate peptide, respectively. We identified 25 novel non-histone peptide substrates of NSD1, but only two of them were (weakly) methylated at the protein level, suggesting that unstructured protein regions are preferred NSD1 substrates. Methylation of H4K20 and p65 which has been described in literature was not observed. We identified methylation of H4K44 (weaker than H3K36) and H1.5 K168, which was much stronger than H3K36 and represents the best NSD1 substrate protein identified so far. Furthermore, we show that Sotos mutations in the SET domain of NSD1 inactivate the enzyme. Our results illustrate the importance of specificity analyses of PKMTs to understand of protein lysine methylation signalling pathways.