N-linked protein glycosylation is an essential and highly conserved post-translational modification in eukaryotes. The transfer of a glycan from a lipid-linked oligosaccharide (LLO) donor to asparagine residues of nascent polypeptide chains is catalyzed by the oligosaccharyltransferase (OST) in the lumen of the endoplasmic reticulum (ER). Trypanosoma brucei encodes three paralogue single protein OSTs called TbSTT3A, TbSTT3B and TbSTT3C that can functionally complement the Saccharomyces cerevisiae OST. We characterized the LLO and polypeptide specificity of all three OST isoforms in the heterologous expression host S. cerevisiae. We demonstrated that TbSTT3A accepted LLO substrates ranging from Man5GlcNAc2 to Man7GlcNAc2. In contrast, TbSTT3B required Man6GlcNAc2 to Glc3Man9GlcNAc2 structures while TbSTT3C did not display any LLO preference. We identified regions within different OST proteins that influence the specificity towards the LLO and polypeptide substrate.