Abstract Giant viruses encode unusual glycosylation machinery distinct from their amoebal hosts, raising fundamental questions about how their glycans are synthesized and diversified. Here, we present a comparative glycomic analysis of mimivirus, tokyovirus and hokutovirus, together with their common host Acanthamoeba castellanii. The main objective of this study was to determine whether giant viruses rely on host-derived N-glycosylation, or alternatively employ virus-encoded pathways to generate lineage-specific O-glycans, and to assess how these processes differ across virus families. N-glycan profiling revealed that all three viruses lack canonical eukaryotic core structures, in contrast to amoebal high-mannose N-glycans carrying pentose and phosphate residues. This finding demonstrates that giant viruses do not exploit the host secretory pathway for N-glycosylation, but instead depend on alternative mechanisms. O-glycan analyses showed lineage-specific patterns: family Marseilleviridae members tokyovirus and hokutovirus, displayed highly similar profiles, with minor virus-specific differences, whereas mimivirus exhibited structurally distinct glycans. Genomic inspection revealed that tokyovirus encodes only five glycosyltransferase-like genes, while A. castellanii harbours candidate enzymes for unusual monosaccharides. These findings clarify the distinct contributions of host and viral pathways and highlight evolutionary diversification of glycosylation among giant viruses.