An 11-year-old, intact female, Yorkshire Terrier dog was presented with epigastric bulging. Results of a CBC included mild neutrophilia and thrombocytopenia. Radiographic examination and abdominal ultrasonography revealed abundant ascites and a well-circumscribed mass in the caudal region of the spleen. Abdominocentesis revealed bloody fluid. Cytologic analysis of the fluid revealed numerous clustered and individual large cells with moderate anisocytosis and anisokaryosis. The spleen was surgically resected. An imprint smear of a white nodular tumor on the caudal pole of the spleen contained a bimorphic population of small and large lymphocytes. The cytologic diagnosis was lymphoma. Histologically, large lymphocytes with distinct borders and single nucleoli formed multiple neoplastic follicles. The final diagnosis was primary splenic lymphoma. Immunocytochemical staining results on buffy coat smears prepared from the ascites fluid showed the lymphocytes were negative for CD3 and positive for CD79a, indicating B-cell origin. Further investigation of the cell clusters using semiquantitative reverse transcriptase-PCR showed that ICAM-1, a cell-cell adhesion molecule, was overexpressed in the tumor cells, likely contributing to the clustering of neoplastic lymphocytes in the ascites fluid. Usually, round cells are not adherent; however, spontaneously detached round cells may form clusters, as in this case, and must be differentiated from epithelial tumors.