In addition to ethanol yield, the production of flavour congeners during fermentation is a major consideration for Scotch whisky producers. Experimental whisky fermentations can provide useful information to the industry, and this is the focus of this paper. This study investigated the impact of wort pretreatments (boiled, autoclaved, filtered) on fermentation performance and flavour development in Scotch whisky distillates as an alternative to freezing wort for storage. Our study showed that no significant sensorial differences were detected in low wines (first distillates), while the chemical compositions showed clear changes in increased levels of esters and higher alcohols in boiled and autoclaved wort. In contrast, filtered wort comprised overall lower levels of congeners. Regarding alcohol yield, all three pretreatments resulted in decreased yields. In practice, the pretreatment of wort prior to fermentation requires additional process operations, while freezing requires large storage units. The pretreatments adopted in this study significantly influence the composition of the malt wort used for experimental whisky fermentations, and this results in a poorer fermentation performance compared with untreated wort. We recommend the use of fresh or frozen wort as the best options for small-scale fermentation trials.