Eukaryotic transcription regulation is dependent on specific histone modifications. Their recognition by epigenetic reader domains triggers complex processes relying on coordinated association of transcription regulatory factors. Although, various modification states of a particular histone residue, e.g. mono-, di- or trimethylation, often lead to differential outcomes, it is not fully understood how they are discriminated at a molecular level. Lens Epithelium Derived Growth Factor/p75 (LEDGF/p75 or PSIP1) is a transcriptional co-activator responsible for tethering other factors to the regions of actively transcribed genes using its PWWP domain that specifically binds di- and trimethylated lysine 36 on histone 3 (H3K36me2/3). Cellular partners bound to its C-terminal integrase-binding domain (IBD) are thus drawn near the active chromatin. Through this interaction, LEDGF/p75 is associated with two distinct diseases, HIV infection and mixed-lineage (MLL) leukemia, and therefore becomes an attractive therapeutic target. Our cryo-EM data capture a LEDGF/p75 PWWP domain in complex with the H3K36 di- and trimethylated nucleosome, respectively. We show that both marks are recognized by the PWWP domain in a highly conserved manner that does not require linker DNA beyond the core nucleosome for stable complex formation even in the absence of chemical crosslinking. Using NMR spectroscopy, we reveal short DNA binding regions in the intrinsically disordered region of LEDGF downstream the PWWP domain that restrain the effective space for assembly of LEDGF-mediated transcriptional complexes. Our data demonstrate that the distinct transcriptional programs triggered by H3K36 di- and trimethylation marks are independent of the actual recognition mode by a cognate reader domain and might require an orthogonal regulatory mechanism.