19Glycosaminoglycans are sulfated polysaccharide molecules, essential for many biological 20 processes. The 6-O sulfation of glycosaminoglycans is carried out by carbohydrate 6-O 21 sulfotransferases (C6OST), previously named Gal/GalNAc/GlcNAc 6-O sulfotransferases. 22Here for the first time we present a detailed phylogenetic reconstruction, analysis of gene 23 synteny conservation and propose an evolutionary scenario for the C6OST family in major 24 vertebrate groups, including mammals, birds, non-avian reptiles, amphibians, lobe-finned 25 fishes, ray-finned fishes, cartilaginous fishes and jawless vertebrates. The C6OST gene 26 expansion likely occurred in the chordate lineage, after the divergence of tunicates and before 27 the emergence of extant vertebrate lineages. 28The two rounds of whole genome duplication in early vertebrate evolution (1R/2R) 29 only contributed two additional C6OST subtype genes, increasing the vertebrate repertoire 30 from four genes to six, divided into two branches. The first branch includes CHST1 and 31 CHST3 as well as a previously unrecognized subtype, CHST16, that was lost in amniotes. 32The second branch includes CHST2, CHST7 and CHST5. Subsequently, local duplications of 33 CHST5 gave rise to CHST4 in the ancestor of tetrapods, and to CHST6 in the ancestor of 34 primates. 35The teleost specific gene duplicates were identified for CHST1, CHST2 and CHST3 36 and are result of whole genome duplication (3R) in the teleost lineage. We could also detect 37 multiple, more recent lineage-specific duplicates. Thus, the repertoire of C6OST genes in 38 vertebrate species has been shaped by different events at several stages during vertebrate 39 evolution, with implications for the evolution of the skeleton, nervous system and cell-cell 40 interactions. 41 42