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Abstract Background Dysregulated T lymphocyte response is thought to play a key role in chronic intestinal inflammation (CIE). Objectives To evaluate the presence of changes in peripheral and intestinal T lymphocyte subsets and to describe potential immune and inflammatory biomarkers in dogs with CIE. Animals Sixteen healthy dogs and 26 dogs were diagnosed with CIE. Methods Prospective case‐control study evaluating peripheral and intestinal T lymphocytes using flow cytometry and inflammatory markers obtained from complete blood cell counts. Results Dogs with CIE had higher peripheral activated T helper (Th) lymphocytes (87/μL [18‐273] CIE, 44/μL [16‐162] healthy control (HC, P = .013) and regulatory T cells (Treg; 108/μL [2‐257] CIE, 34/μL [1‐114] HC, P = .004). In the intestinal epithelium, CIE dogs presented lower percentages of Th (4.55% [1.75‐18.67] CIE, 8.77% [3.79‐25.03] HC, P = .002), activated Th cells (0.16% [0.02‐0.83] CIE, 0.33% [0.05‐0.57] HC, P = .03) and CD4/CD8 ratio (0.08 [0.02‐0.39] CIE, 0.21 [0.07‐0.85] HC, P = .003). Conversely, higher percentage of activated T cytotoxic cells (20.24% [3.12‐77.12] CIE, 12.32% [1.21‐39.22] HC, P = .04) and interferon‐gamma (IFN‐γ) producing T lymphocytes (7.36% [0.63‐55.83] CIE, 1.44% [0.00‐10.56] HC, P = .01) within the epithelium was observed. In the lamina propria the percentage of Treg lymphocytes was higher (6.02% [1.00‐21.48] CIE, 3.52% [0.18‐10.52] HC, P = .02). Conclusions and Clinical Importance Systemic and intestinal immune alterations occur in dogs with CIE suggesting that blood IFN‐γ producing T lymphocytes and the systemic immune‐inflamation index (SII) could potentially serve as biomarkers for the disease.