Chlamydomonas reinhardtii (C. reinhardtii) N- glycans carry plant typical β1,2-core xylose, α1,3-fucose residues, as well as plant atypical terminal β1,4-xylose and methylated mannoses. In a recent study, XylT1A was shown to act as core xylosyltransferase, whereby its action was of importance for an inhibition of excessive Man1A dependent trimming. N- Glycans found in a XylT1A/Man1A double mutant carried core xylose residues, suggesting the existence of a second core xylosyltransferase in C. reinhardtii . To further elucidate enzymes important for N -glycosylation, novel single knockdown mutants of candidate genes involved in the N -glycosylation pathway were characterized. In addition, double, triple, and quadruple mutants affecting already known N -glycosylation pathway genes were generated. By characterizing N -glycan compositions of intact N -glycopeptides from these mutant strains by mass spectrometry, a candidate gene encoding for a second putative core xylosyltransferase (XylT1B) was identified. Additionally, the role of a putative fucosyltransferase was revealed. Mutant strains with knockdown of both xylosyltransferases and the fucosyltransferase resulted in the formation of N- glycans with strongly diminished core modifications. Thus, the mutant strains generated will pave the way for further investigations on how single N- glycan core epitopes modulate protein function in C. reinhardtii .