Aotus monkeys offer one of the few models that can be used for the evaluation of the immunogenicity and efficacy of new vaccine candidates against the human malarias, Plasmodium falciparum and Plasmodium vivax. However, the tools available for evaluation of the immune responses in these New World primates are still limited. In the present study, a previously selected set of monoclonal antibodies that were raised against human T cell determinants and were reactive with at least one other primate species was investigated for its reactivity with Aotus lymphocytes using FACS analysis, indirect immunofluorescence (IFA) and immunohistochemistry. From a panel of 19 mAb, six were found to react consistently with Aotus lymphocytes using FACS analysis. Further evaluation of the mAb using IFA confirmed these findings. Analysis of the selected mAb on spleen sections of Aotus monkeys identified one anti-CD4 and one anti-CD8 mAb that can be used for immunohistochemical studies. The set of mAb identified in this study can be used for the detection of various T lymphocyte markers in peripheral blood and in tissues of Aotus monkeys. Together with data published by others, mAb are now identified for detection of six different markers of Aotus T lymphocytes. These mAb are very valuable for the characterisation of immune responses after vaccination and infection in the Aotus malaria models.