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Visceral leishmaniasis (VL) is a disease causing several clinical manifestations in dogs, including neurological disorders. Nevertheless, there are few studies related to the evaluation of the brain alterations during VL. Evidences of the involvement of cerebral barriers in infected dogs was reported, including the presence of brain inflammatory infiltrate, with a predominance of CD3+ T cells. Therefore, the aim of this study was to determine the immunophenotypes of T lymphocytes in the cerebrospinal fluid (CSF), as well as in peripheral blood, and to correlate with brain alterations in dogs with VL. We detected elevated percentages of double negative (DN) and double positive (DP) T cells in the CSF, with a predominance of TCRαb. In the histopathological analysis, we observed a predominance of lymphoplasmacytic infiltrate, mainly in leptomeninges, ranging from mild to intense, and we observed a positive correlation between the intensity of inflammation in the subependymal area and the DN T cells of the CSF. Thus, the DN T cells seem be acting as villains of the immune system through pro-inflammatory mechanisms. Further, the proportion of the different population of CSF T cells did not differ from those observed in the blood, which provides us with more evidence of blood-CSF barrier breakdown. Together, the results provide more explanation to the inflammation observed in the brain of dogs with VL, which the DN T cells contribute to the origin and progression of the neurological disease. This study provides insight into the immunophenotypes of T lymphocytes in the CSF during canine visceral leishmaniasis.