Species cross-reactive anti-peptide antibodies were assessed in formalin-fixed tissue for use in immunophenotyping of lymphosarcoma in two alpacas. Diagnosis of lymphosarcoma was made by routine histopathological examination. Primary antibodies used for immunophenotyping were anti-human CD3 and anti-human CD5 for T cells; and anti-human CD79a and anti-human CD79b for B cells/plasma cells. In one case, most of the neoplastic cells were labelled with both anti-CD3 and anti-CD79b, and smaller numbers were labelled with anti-CD79a. The other case was classified as a B-cell tumour on the basis of labelling of the majority of neoplastic cells with anti-CD79b and anti-CD79a. This is the first recorded attempt at immunophenotyping lymphosarcoma in alpacas and, to our knowledge, the first record of presumptive co-expression of T- and B-cell-associated molecules in lymphosarcoma in the veterinary literature.