<span class="paragraphSection"><div class="boxTitle">Abstract</div>Histone variants along with their associated chaperones have been considered as one of the major complexes to provide versatility in organizing chromatin structure. Post-translational modifications (PTMs) of H3 variants serve as very important factors in promoting heterochromatin assembly, protecting telomere stability, and suppressing transposon activity. However, the precise mechanism by which specific PTMs on H3 variants suppress transposons remains elusive. Here, by monitoring retrotransposon mobilization during <span style="font-style: italic;">Drosophila</span> hindgut development, we identified the DNA synthesis-coupled (DSC) H3.2K9me2 deposition pathway as a pivotal mechanism for transposon suppression. Depleting the factors in the DSC H3.2 complex, but not in the DNA synthesis-independent (DSI) H3.3 chaperone pathway, unleashed massive retrotransposon activation. DSC chaperones specifically establish dimethylation at the H3.2K9 site in heterochromatic regions by directly interacting with and recruiting the histone methyltransferase, G9a. Intriguingly, the cross-talk between DSC H3.2K9me2 and DSI H3.3K9me3 in heterochromatin is dynamically regulated and properly balanced. Although DSI H3.3K9me3 could efficiently be incorporated into transposon loci when the DSC H3.2K9me2 deposition pathway was disrupted, H3.3K9me3 alone was insufficient to establish functional heterochromatin required for transposon silencing during development. Altogether, our discoveries provide a framework to understand how cells employ specific histone variant modifications to construct and maintain heterochromatin, thereby ensuring transposon repression and safeguarding genome integrity.</span>