Post translational modifications (PTM) enhance the functional diversity of proteins. Among these PTM, glycosylation represents one of the most abundant and complex modification of proteins and N-glycosylation is determinant for cell survival. For 96% of N-glycosylproteins, glycosylation occurs at the canonical motif [N]-!P-[S/T] known as sequon [1]. In the laboratory, we study the evolutionary relationships and molecular evolution of glycosyltransferases that are Golgi enzymes implicated in the last steps of glycosylation [2] like B4GALNT2 implicated in the synthesis of blood group antigen Sda [3]. This enzyme is a glycoprotein and our working hypothesis is that the gain or loss of an ancestrally conserved N-glycosylation site in this enzyme might have resulted in the evolution of protein structure, subcellular localization and functional modifications with impact on the phenotype [4-7]. As a first towards answering this question, we identified over 150 B4GALNT-related sequences (37 B4GALNT1 paralogues and 85 B4GALNT2 orthologues) from public databases (NCBI, Ensembl, CAZy) using a BLAST approach. We carried out sequence-based analysis using various multiple sequence alignment algorithms (MUSCLE, ClustalΩ, MAFFT, T-Coffee, ClustalW) to delineate informative regions and define conserved sequence motifs. Furthermore, we used NetNGlyc 1.0 server [8] and GlycoMine [9] to predict potential N-glycosylation sites. We found differentially conserved N-glycosylation sites in the homologous B4GALNT sequences: a canonical sequon, [N]-!P-[S/T], present in 11 mammalian B4GALNT2 sequences, which has disappeared in the human B4GALNT2 sequence [10]. This N-glycosylation site is also present in some ancestral B4GALNT sequences and has disappeared in the paralogous B4GALNT1 sequences suggesting no major role for this N-glycan. Interestingly, two atypical N-glycosylation sites [N]-X-[C] and [N]-[G] were predicted in mammalian B4GALNT2 sequences. Despite the fact that the [N]-[G] site is present in almost all the mammalian B4GALNT2 sequences, we found using PNGase F, an enzyme that remove N-Glycans, that the human B4GALNT2 enzyme is not glycosylated at this position, but at a second unusual [N]-X-[C] glycosylation site. This latter was found to be highly conserved in nearly all B4GALNT sequences except the teleostean B4GALNT2 sequences further suggesting an important functional role, which is currently investigated.