The combination of RAFT chemistry and hetero-Diels-Alder (HDA) cycloaddition provides a convenient conjugation tool for the construction of macromolecular architectures. The actual conjugation linkage, which is formed in the RAFT HDA approach, consists of a 3,6-dihydro-2H-thiopyran ring. Herein, a careful study into the stability of polymer linked by 3,6-dihydro-2H-thiopyran rings under different thermal and pH conditions is performed. As polymeric model system, a 3,6-dihydro-2H-thiopyran functionalized poly(ethylene glycol) (PEG) obtained after a HDA cycloaddition between a diene functionalized PEG and 1-phenylethyl (diethoxyphosphoryl)dithioformate or 1-phenylethyl pyridin-2-yldithioformate is used. Electrospray ionization mass spectrometry (ESI-MS) is employed to map the generated products. The stability tests under thermal conditions showed for the diethoxyphosphoryl derivative the preservation of the polymer end-groups after 24 h at temperatures up to 80°C. At higher temperatures degradation reactions of the 3,6-dihydro-2H-thiopyran ring including the retro HDA reaction occurred. A complete fragmentation was observed after 24 h at 160°C. The pyridinyl derivative was found to be slightly more temperature stable since its structure was unaffected at conditions up to 24 h at 120°C and full cleavage was only observed after 24 h at 180°C. In this case the fragmentation mechanism occurred exclusively via the retro HDA reaction and the resulting diene functionalized polymer was the only detected product species. Both conjugates showed an excellent stability toward hydrolysis in aqueous media under strong acidic (0.1 and 1 M HCL) and strong basic (0.1 and 1 M NaOH) conditions.