Ploidy is a fundamental genetic concept that refers to the number of sets of chromosomes in an organism’s cells. Most salmonids are naturally diploid, possessing two sets of chromosomes, but triploid salmon are increasingly produced through breeding techniques to enhance specific traits. Triploid salmonids, which have three sets of chromosomes, demonstrate accelerated growth rates and improved disease resistance, making them particularly valuable in aquaculture. While the triploidization of salmonids offers several advantages, it also presents challenges and concerns. A significant issue is the potential reduction in genetic diversity, reduced adaptability and resilience to environmental changes, and diseases. Studies have shown that triploid Chinook salmon exhibit 10-30% higher mortality rates and increased susceptibility to diseases compared to their diploid counterparts under aquaculture conditions. Chinook salmon, the largest and most valued species of Pacific salmon in North America, are prized for their size, resilience, flavor, and nutritional composition, making them an attractive candidate for diversifying Canadian aquaculture. Consequently, we investigated the impact of triploidization on the gut of Chinook salmon, an organ vital for their overall health and survival. We also evaluated how molecular and structural markers are modulated in each ploidy during an infection challenge using Vibrio anguillarum. To achieve this, we assessed the expression of genes associated with the innate and adaptive immune responses, as well as gut barrier integrity, in the pyloric gut, midgut, and hindgut of both diploid and triploid Chinook salmon. Additionally, we measured protein levels of key immune markers through Western blot and ELISA. Histological analysis was conducted to determine the structural effects of triploidization on the gut. Our results indicate that triploidization influences the modulation of genes across different gut sections, as well as the response of each section to V. anguillarum challenge. Notable differences were observed in the levels of four proteins—β2m, IL-1β, IgM, and IgT—between the ploidies, with particularly interesting findings of consistently higher β2m levels in infected triploids compared to diploids. We anticipate that the histological findings will help clarify whether these molecular variations between ploidies correspond to structural changes in the Chinook salmon gut or are confined to molecular