Very recently, we identified two distinct mesenchymal stem cell (MSC) subsets in primary bone marrow (BM) that differ in their expression pattern (CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-)) and morphology as well as in their clonogenic and differentiation capacity. Here we analyzed the cell surface antigen expression in these subsets in more detail and compared the profiles with the expression pattern on cultured MSCs. Most of the tested antigens, including CD13, CD15, CD73, CD140b, CD144, CD146, and CD164, are expressed at similar levels in both primary BM populations. However, a number of markers were differentially expressed. Of these, CD166 (ALCAM), CD200, and CD106 (VCAM-1) showed an almost selective expression on either CD271(bright)MSCA-1(dim)CD56(+) (increased CD166 and CD200 expression) or CD271(bright)MSCA-1(bright)CD56(-) (increased CD106 expression) MSCs, respectively. Additional markers with elevated expression on CD56(+) MSCs include F9-3C2F1, HEK-3D3, HEK5-1B3, and W1C3 antigens, whereas CD10, CD26, CD106, 7C5G1, 9A3G2, 56A1C2, 66E2D11, HEK-3D6, HEK4-1A1, HEK4-2D6, W1D6, W4A5, W7C6, and W8B2 (MSCA-1) antigens showed increased expression in the CD56(-) population. The majority of the analyzed markers found on primary MSCs were also expressed on cultured MSCs. However, in contrast to primary MSCs, HEK7-1C4, W1C3, W1D6, and W4A5 antigens were absent on the cultured counterparts. 7G5G1 and 9A3G2 antigens showed reduced, and HEK-3D6, F9-3C2, and HEK-3D3 showed increased expression on cultured cells. The extended knowledge about the phenotype of the two subsets and the identification of novel MSC markers may result in the isolation of attractive starting populations for applications in regenerative medicine.