While human B cells have been extensively studied, most reports have used peripheral blood as a source. Here we have used a unique tissue resource derived from healthy organ donors to deeply characterize human B cell compartments across multiple tissues and donors. These data sets revealed that B cells in the blood are not in homeostasis with compartments in other tissues. We found striking donor-to-donor variability in the frequencies and isotype of CD27+ memory B cells (MBC). A comprehensive Ab-based screen revealed markers of MBC and allowed identification of novel MBC subsets with distinct function defined by surface expression of CD69 and CD45RB. We defined a tissue-resident MBC phenotype that was predominant in the gut but absent in blood. RNA sequencing of MBC subsets from multiple tissues revealed a tissue-resident MBC gene signature as well as gut and spleen-specific signatures. Overall, these studies provide novel insights into the nature and function of human B cell compartments across multiple tissues.