Canine atopic dermatitis (CAD) is a chronic and inflammatory skin condition with a multifaceted origin, involving genetic factors, skin barrier abnormalities, immune responses, and hypersensitivity to various allergens. Interleukin 33 (IL-33), released by keratinocytes upon cellular injury, plays a crucial role in atopic dermatitis pathogenesis by inducing Th2 lymphocyte-mediated immune responses. This study aimed to evaluate IL-33 expression in dogs with atopic dermatitis and compare it to a control group. Forty-nine dogs were included, with 39 having atopic dermatitis, subdivided into groups based on clinical characteristics, and ten in the control group. Lesion and pruritus scores were assessed, and incisional biopsies were analyzed for dermatopathological characteristics. IL-33 expression was evaluated using immunohistochemistry, the analyses were blinded, based on the measurement of immunostaining areas using Image Pro-Plus software, version 4.5, relying on a semi-automatic color segmentation method, where the tissue immunostaining area for each biomarker was artificially delimited and quantified. Statistically significant differences in IL-33 immunostaining were found among groups (P=0.0005). Lichenified dogs (group 4) exhibited higher immunostaining compared to erythema (group 3) (P=0.0006), alesional pruritus (group 2) (P=0.0261), and the control group (group 1) (P=0.0079). IL-33 immunostaining increased with lesion progression, strongly correlating with lesion scores (P<0.0001), particularly in patients with chronic lesions characterized by erythema and lichenification. These findings suggest IL-33's significant role in canine atopic dermatitis pathogenesis and its association with lesion and inflammation scores during the chronic phase. This suggests potential therapeutic interventions targeting IL-33 or its receptors, though further studies are needed to explore these possibilities.