Glycosylation, the chemical linkage of carbohydrates to proteins, is a common post-translational modification within bacteria known to influence virulence. Within the Burkholderia genus multiple species can cause opportunistic human infections and O-linked glycosylation is required for virulence in at least two pathogenic species, Burkholderia cenocepacia and Burkholderia pseudomallei. Across the Burkholderia genus O‑linked glycosylation is highly conserved, with the O‑linked glycosylation cluster required for the generation of the trisaccharide glycans used for glycosylation, and the O-oligosaccharyltransferase PglL, responsible for the transfer of these trisaccharides to protein substrates. Within all Burkholderia species examined to date, more than 100 proteins are subjected to glycosylation. Many of these glycoproteins are conserved across the Burkholderia genus, yet most have no known function. To investigate the conserved role of glycosylation, we have used CRISPR interference (CRISPRi) to target pglL, enabling the inducible silencing of glycosylation across several Burkholderia species. Validating this approach in B. cenocepacia, we demonstrate that CRISPRi silencing results in the suppression of pglL and leading to a >88% reduction in glycosylation (as determined by glycopeptide analysis). In line with the reduced glycosylation, CRISPRi silencing results in similar proteome changes observed in pglL mutant strain, yet only partially phenocopies known defects observed this glycosylation mutant. Excitingly, this suggests reductions in glycosylation are differently tolerated across the glycoproteome. Expanding this CRISPRi approach to B. thailandensis, B. diffusa, and B. multivorans has now allowed us to compare the impact on glycosylation silencing between different Burkholderia species. This comparative proteomic and glycoproteomic analysis reveals glycosylation inhibition results in similar impacts on the proteome that are associated with membrane stress and determine that multiple shared glycoproteins across the genus are sensitive to changes in glycosylation levels. Combined, this work demonstrates CRIPSRi silencing provides a robust means to modulate glycosylation levels across bacterial species and is a powerful tool for probing the impact of glycosylation across bacterial species.