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Agar polysaccharides extracted from two Thai species of Gracilaria (G. fisheri and G. edulis) and one Japanese species (Gracilaria sp.) were investigated by physical and chemical analysis, and 1 H, 13 C NMR and FT-IR spectroscopy. Agar with partial 6-O-methylated on 3-linked β-D-galactopyranosyl, 2-O-methylated on 4-linked 3,6-anhydro-α-L-galactopyranosyl and 4-O-methyl-α-L-galactopyranosyl units attached to the C6 of 3-linked D-galactopyranosyl units were isolated from G. fisheri. The large parts of 6-O-methylated on 3-linked β-D-galactopyranosyl units and partial methylation on C2 of 4-linked 3,6-anhydro-α-Lgalactopyranosyl units were observed in the agar extracted from G. edulis which corresponded with higher gelling temperature (»60 C°). In contrast, the Japanese agar extracted from Gracilaria sp. showed a typical pattern of agarobiose with partial methylation at C6 of the D-galactopyranosyl units. All agars extracted exhibited sulfate substitution at different positions in the polymers. High sulfate contents were obtained in native agar of G. fisheri (4.56%) and G. edulis (7.54%) that mainly branched at C-4 of the D-galactopyranosyl unit. The presence of this unit was responsible for poor gelling ability of the agar polymers. Alkali treatment was effective both in removing alkali-labile sulfate and increasing the gel strength in Gracilaria sp. (334.5 ± 14.1 g/cm 2) whereas only a slight effect was noted on G. fisheri (228.27 ± 48.18 g/cm 2) and G. edulis (239.95 ± 28.35 g/cm 2). Further investigation may need to determine the constituent sugars and an alternative utilization of the Thai gracilaroids.