In aquaculture, fish are exposed to a range of unfavourable environmental conditions. Amongst these, stocking density has attracted considerable attention as inappropriate densities may compromise welfare and negatively impact production. However, the recommendations for stocking remain elusive. The aim of the present study was to apply a novel method to investigate a level of crowding that indicated aversiveness in rainbow trout (Oncorhynchus mykiss). In a two-tank system, where two identical tanks were connected via a doorway, it was observed that social behaviour controlled the distribution of the fish between the tanks. Fish were stocked at equal quantities in each tank of the system. The doorway was opened and the fish moved between the two tanks. Typically, this resulted in one tank being occupied by a few highly aggressive dominant individuals (“dominant” tank) and the majority of the fish occupying the second tank (“crowded” tank). Here, the potential of this unequal spatial distribution for quantifying aversion to crowding was explored. Fish were stocked in three two-tank systems at a total density of 20, 40 and 80kgm−3 respectively. The number of fish in each tank was determined every three days throughout the duration of the experiment and the percentage of fish in the “crowded” tank was used as an indicator of the distribution pattern in the two-tank systems. The results indicated a negative relationship between the total density stocked (20, 40 and 80kgm−3) and the percentage of fish in the “crowded” tank. A subsample of individuals was sacrificed for blood and brain samples every three days from the “crowded” tank, prior to the fish count. The neuroendocrine indicators of stress, elevated serotonergic activity levels which were not associated with high plasma levels of cortisol, suggested chronic stress in the fish at the highest total density stocked (80kgm−3). Taken together, these results indicated that a level of aversiveness to crowding had been reached at the highest density stocked, where the mean absolute density, irrespective of time of day, observed in the “crowded” tank was 126.5±3.7kgm−3.