This study is related to the post-crosslinking of thiolated polyvinyl alcohol (TPVA) hydrogels using three crosslinkers, sodium trimetaphosphate (STMP), boric acid (BA) and glyoxal (GLY) under alkaline conditions. The crosslinking reaction was carried out under different conditions: crosslinker nature, crosslinker content and crosslinking time. The influence of different crosslinkers on the physicochemical and structural characteristics of TPVA was evaluated. The three reagents used for crosslinking presented different action mode on hydrogels. The optimized crosslinking conditions were the crosslinker content is 0.1 % (w/v) at crosslinking time of 30 min. The results showed that the STMP-crosslinked TPVA had the maximum thiol content and swelling ability. All crosslinked TPVA samples were stable in different pH media. The involvement of thiol and other relevant groups after crosslinking in the different crosslinked TPVA samples was confirmed by attenuated total reflectance-infrared spectra. The microanalysis of elements present in the crosslinked samples were analyzed by energy-dispersive X-ray microanalysis.