In this paper, the involvement of iso-α-acids in the appearance of stale flavour in beer during storage was examined. Flavour instability upon storage, by far the most important quality problem of beer, may be caused by a multitude of reactions, in particular, the degradation of trans-iso-α-acids is pivotal. In order to gain improved understanding of the beer ageing process, the behaviour of the stereoisomers of the bitter acids was studied for the first time in pilot beers at particularly high and low proportions of the trans-iso-α-acids concentration relative to the cis-iso-α-acids concentration. Therefore, pure trans- respectively cis-isomers were required and obtained on a pilot scale by the separation of trans-iso-α-acids as β-cyclodextrin inclusion complexes from a commercial isomerised hop extract, permitting the quantitative dosage to beer in mg/L amounts. The relationship between the storage-induced degradation of these iso-α-acids in authentic beer samples and the increase in staling aldehydes, especially 2-methylpropanal, 2-methylbutanal and 3-methylbutanal, is the subject of further investigations. A remarkable increase in the relative concentration of cis-iso-α-acids was observed in the beers bittered with purified trans-iso-α-acids, which may be ascribed to partial conversion, i.e., reverse isomerisation, of trans-iso-α-acids via α-acids into cis-iso-α-acids. In spite of the trans-specific degradation observed during ageing, the decomposition of these bitter acids leading to volatile carbonyl compounds is of minor importance. Aldehyde formation as a function of forced ageing was irrespective of the mode of bittering, emphasising that malt quality and the brewing process itself are probably the most important factors regarding the flavour instability of beer.