Structural analysis of the two TRIM33 isoforms shows that the 17 amino acid insertion in TRIM33α, relative to TRIM33β, results in a change in orientation for N1039. This Asn residue, which is equivalent to N140 in BRD4(1), typically forms hydrogen bonding interactions with KAc, and is conserved amongst the canonical BRDs. This difference gives rise to the possibility that the BRDs of TRIM33α and TRIM33β function differently, with the TRIM33α BRD not binding to KAc, and therefore not recruiting this protein to chromatin. Establishing an AlphaScreen assay allowed us to probe which post-translations modifications (PTMs) on a histone 3-mimicking peptide (H31-27) promote binding to TRIM33α and TRIM33β. We show that both isoforms of TRIM33 bind preferentially to H31-27K18Ac over other acetylated peptides, and computational studies show that this preference results from an interaction between H3R17 and E981 of TRIM33. H3K14 and H3K23 have different flanking amino acids, meaning that this interaction does not form. Interestingly, H3K9 and H3K27 are both preceded by an Arg residue, but we did not investigate the effect of acetylation at these positions on TRIM33 BRD binding.