Traditional sparkling wines are obtained from a vinification process with an additional ‘second’ fermentation in the bottle, which has an essential impact on the sensorial characteristics. During this maturation phase, several compounds are released from the yeast in autolysis, including the tripeptide glutathione. This compound has recently gained more interest due to its role in the formation of polysulfides and the subsequent possibility of H2S release post‐bottling. Hence, the release of sulphur‐containing compounds like glutathione during an ageing ‘sur lies’ ought to be considered regarding the accumulation and persistence of polysulfides. In this study, the presence of polysulfides in traditional sparkling wines was validated to gain a preliminary understanding of the role of ageing time and glutathione. UHPLC‐HRMS allowed the detection of two glutathionyl trisulfides in a selection of different naturally sparkling wines. Besides, it was found that the wines from vintage 2020 had a relatively high formation of GS3G and GS3C compared to vintages 2018 and 2019. Moreover, a trend between the trisulfides and GSSC was observed, possibly related to glutathione condensation and cysteine uptake by the yeast. This study showed the presence of polysulfides in various sparkling wines for the first time, increasing the availability of the current knowledge on this topic.