Introduction: Investigations in murine models have identified myocardial B cells with a characteristic gene expression signature and have pointed at B cells as a promising target to develop novel therapies for heart failure. However, there is almost no data on B cells in the human heart. Methods and Results: We performed immunostaining to characterize the amount and distribution of B cells in human hearts. In 12 healthy hearts collected at autopsy, we found 1.4 ± 1.6 CD20+ B cells per high power field. The number of CD3+ T cells was almost 20 times higher (26.8 ± 28.6, P<0.001). Utilizing CD31 (endothelial marker), we found that human myocardial B cells were both in intravascular and interstitial locations, with a slight prevalence for the interstitium (P=0.006). Parallel analysis of tissue collected post-mortem or at the time of VAD placement showed that analysis of post-mortem tissue did not introduce bias. To gain insight into the biology of human myocardial B cells we first analyzed publicly available single cell sequencing datasets of myocardial (Heart Cell Atlas) and peripheral blood cells (10X Genomics). We found that the human myocardium harbored a minor population of B1 cells and a larger population of B2 cells, and that myocardial B cells had a gene expression signature distinct from that of peripheral blood B cells. We validated these findings on primary B cells sorted from the heart and peripheral blood of 2 patients subjected to LVAD implant (binomial for concordance p=4.13e-32). To identify biological pathways upregulated in myocardial B cells across species, we compared differential gene expression in myocardial vs peripheral blood B cells across the studied human datasets and published rodent datasets. We identified 703 genes with consistent differential gene expression (binomial p=2.9e-48). KEGG pathway analysis of these genes highlighted “B cell receptor signaling pathway”, “Antigen processing and presentation” and “Cytokine-cytokine receptor interaction” among the top conserved pathways upregulated in cardiac B cells (FDR <0.001). Conclusions: The human heart harbors a small population of B cells that is comprised of both B1 and B2 cells. Human myocardial B cells have a distinct gene expression signature that is conserved across species.