Lymphoma is an important disease in dogs and people, with similar biological characteristics. We tested the binding affinity of a peptidomimetic LLP2A, previously shown to bind the alpha4-beta1 integrin on human lymphoma cell lines, to lymphocytes of dogs with spontaneously occurring lymphoma. Fine needle aspirates of lymph nodes from 32 dogs with B-cell lymphoma and 7 dogs with T-cell lymphoma were evaluated using flow cytometry. For B cells, the lowest MFI levels were in unlabeled, non-neoplastic lymphocytes. The highest median fluorescent intensity (MFI) levels occurred in LLP2A-labeled lymphoma cells from dogs that had not received chemotherapy followed by labeled lymphoma cells from dogs that had received chemotherapy. The fluorescence profile of the T-cell samples was similar although many of the differences were not statistically significant, likely due to low sample number. Specifically, LLP2A-labeled T-cell lymphoma cells had a significantly higher MFI compared to unlabeled non-neoplastic lymphocytes. LLP2A affinity was not significantly different in unlabeled and labeled T-cell lymphoma cells, and labeled non-neoplastic lymphocytes. For both B and T cells, labeling with LLP2A tended to increase MFI in both normal and lymphoma cells. Lymphoma cells had higher mean MFI levels than non-neoplastic lymphocytes, and chemotherapy acted to decrease MFI. In summary, these data demonstrate that LLP2A has affinity to canine lymphoma cells and indicates expression of the alpha4-beta1 integrin on these cells. In fact, LLP2A preferentially binds neoplastic B-cells, suggesting that this small molecule may be of use in cross-species clinical trials of targeted therapeutics.