We also show that both isoforms of TRIM33 bind to H31-27K9Me3. Affinity for these two PTMs is additive, and structural studies show that these marks are suitably placed to enable simultaneous binding in cis. Studies using the W889A mutation in the TRIM33 PHD shows this residue is important for H31-27K9Me3 binding in both isoforms. However, the N1039F mutation in the BRD only affected binding of K18Ac to TRIM33β but not TRIM33α, consistent with the idea that N1039 is not involved in histone peptide binding in this isoform. It is interesting, however, that despite the difference in N1039 orientation, both TRIM33α and TRIM33β show similar affinities for H31-27K18Ac. This is consistent with the idea that this residue helps to direct KAc into the pocket, but interactions with N1039 do not contribute substantially to the affinity of the BRD for KAc. Computational studies indicate that the affinity of the histone 3-mimicking peptide for TRIM33α results in part from hydrophobic interactions between H3K18Ac and F1038, which do not occur in TRIM33β. Unexpectedly, this suggests that, despite their structural differences, TRIM33α and TRIM33β can both bind to similar modifications on histone 3, indicating that both isoforms could be involved in locating TRIM33 to sites of DNA damage. This finding has implications for future design of ligands for the TRIM33 BRD and BRDs more generally. Analysis of a range of cancer and non-cancer cell lines shows that TRIM33 has higher expression levels in cancer cell lines. Its role in DNA damage response means that inhibition of the function of the BRD, PHD, or both, could be a therapeutic target in oncology. To identify leads to progress this work, we identified four new ligands for the TRIM33 PHD-BRD cassette and show that one of these binds to BRD. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33, and which residues are important in this process. Design of TRIM33 PHD and BRD ligands can build on these observations, potentially using different strategies to those taken for other BRDs. Given that TRIM33 possesses E3 ligase activity, the identification of ligands for its BRD raises the possibility that PROTACs that recruit TRIM33 to degrade a protein of interest can be developed. It remains to be seen whether ligands that are selective for TRIM33α or TRIM33β can be developed, and whether they have different activity, either individually or as an E3 ligase ligand in PROTACs. The data that we report here paves the way for such investigations.