•57R2A, a novel monoclonal antibody against mouse GPR56, was established.•Flow cytometry using 57R2A demonstrated GPR56 expression in the mouse hematopoietic stem cell (HSC) fraction.•57R2A marked long-term repopulating (LTR) cells in mouse bone marrow mononuclear cells (BMMNCs).•LTR potency was consistent with the expression of GPR56 and MPL.•Findings for 57R2A suggest that GPR56 is a positive marker for mouse HSCs. GPR56 molecule, a G-protein-coupled receptor, was suggested to be expressed in mouse hematopoietic stem cells (HSCs) by gene expression analyses. However, little is known about the cell surface expression of GPR56 protein in mouse HSCs due to the absence of an appropriate monoclonal antibody against GPR56 for flow cytometry analyses. In the present study, we established a novel monoclonal antibody against mouse GPR56 (57R2A) to examine the expression and distribution of GPR56 protein in HSCs. A flow cytometry analysis using 57R2A showed that GPR56 was highly expressed in the CD34−, c-Kit+, Sca-1+, lineage-negative (Lin−) fraction, which are highly enriched with HSCs. The competitive long-term repopulation (LTR) assay showed that LTR cells were included only within the GPR56+ fraction (≤15%) of bone marrow mononuclear cells (BMMNCs), but not within the remaining GPR56− fraction (85%), suggesting that all HSCs express GPR56 protein on their surface. Furthermore, we showed that double staining of BMMNCs with only 57R2A and AMM2 (monoclonal antibody against the HSC marker MPL) enabled enrichment of all LTR cells in the double-positive fraction (0.8% of BMMNCs), within which the LTR potency was consistent with the expression of both GPR56 and MPL. In conclusion, these findings for 57R2A suggest that all HSCs in mouse BMMNCs express GPR56 protein on their surface and that GPR56 is a positive marker for HSCs.