The ability to elicit potent and long-lasting broadly neutralizing HIV envelope (Env)-specific antibodies has become a key goal for HIV vaccine development. Consequently, the ability to rapidly and efficiently monitor development of memory B cells in pre-clinical and clinical vaccine trails is critical for continued progress in vaccine design. We have developed an improved flow cytometry-based method for the rapid and efficient identification of gp120-specific memory B cells in peripheral blood, bone marrow, and mucosal tissues which allows their direct staining without the need for prior cell sorting or enrichment. We demonstrate staining of both HIV and SIV Env-specific memory B cells in PBMC, bone marrow, and rectal tissue of vaccinated and infected rhesus macaques. Validation of the method is illustrated by statistically significant correlations with memory B cell levels quantified by ELISPOT assay and with serum binding antibody titers determined by ELISA. In addition to quantification, this method will bring the power of flow cytometry to the study of homing and trafficking of Env-specific memory B cells.