Lymphocytosis caused by neoplastic proliferation of small lymphocytes is occasionally difficult to distinguish by morphological examination from nonneoplastic lymphocytosis. To examine the clinical utility of gene rearrangement analysis for demonstrating neoplastic proliferation of small lymphocytes, gene rearrangement analysis was performed in comparison with immunophenotyping using peripheral lymphocytes in dogs with small lymphocytosis. Thirty-one dogs with small-cell lymphocytosis (8,100-884,300/microl) were enrolled. By immunophenotyping, lymphocytosis of all dogs was suggested to be neoplastic in nature based on the detection of marked expansion of phenotypically homogeneous lymphocytes or the presence of an aberrant antigen-expressing population of lymphocytes. In contrast, gene rearrangement analysis represented clonality in 27 dogs (detection rate of 87%). From the present study, gene rearrangement analysis was considered to be worthwhile to strengthen the evidence of neoplastic proliferation of small lymphocytes when coupled with immunophenotyping and to be a suitable diagnostic substitute if immunophenotyping is not available in clinical practice.