Rhesus macaques (Macaca mulatta) are used as a human-relevant animal species for the evaluation of vaccines and as a source for cloning monoclonal antibodies (mAbs) that are highly similar to human-derived antibodies. Although antibody-secreting plasmablasts in humans are well-defined and can be easily isolated for mAb cloning, it remains unclear whether the same phenotypic markers could be applied for isolating antibody-secreting plasmablasts from Chinese rhesus macaques. In this study, we evaluated a series of cell surface and intracellular markers and identified the phenotypic markers of plasmablasts in Chinese rhesus macaques as CD3-CD14-CD56-CD19-CD27-CD20-/lowCD80+HLA-DR+CD95+. After influenza virus vaccination, the plasmablasts in peripheral blood mononuclear cells (PBMCs) increased transiently, peaked at day 4-7 after booster vaccination and returned to nearly undetectable levels by day 14. Antigen-specific enzyme-linked immunosorbent spot (ELISPOT) assays confirmed that the majority of the plasmablasts could produce influenza virus-specific antibodies. These plasmablasts showed transcriptional characteristics similar to those of human plasmablasts. Using single-cell PCR for immunoglobulin heavy and light chains, most mAbs cloned from the CD3-CD14-CD56-CD19-CD27-CD20-/lowCD80+HLA-DR+CD95+ plasmablasts after vaccination exhibited specific binding to influenza virus. This study defined the phenotypic markers for isolating antibody-secreting plasmablasts from Chinese rhesus macaques, which has implications for efficient cloning of mAbs and for the evaluation of plasmablast response after vaccination or infection in Chinese rhesus macaques.