Mesenchymal stromal cells (MSCs) established by in-vitro adherence culture have been widely utilized for various cell therapeutic trials, but potential heterogeneity that can be caused by preparation methods are poorly characterized. In this study, we show that at least two distinct subsets of MSCs with different adherence to plastic surface exist in human adipose tissue-derived stromal vascular fraction (SVF); while 69% of total colony forming units in SVF adhere to the surface before 3 hrs of plating, 13–17% of colonogenic cells adhered to the surface at later period of 15 hr to 1 week after plating. Of note, the late adherent MSCs exhibited higher self-renewal of colony forming cells and higher proliferating potential with comparable level of osteogenic or adipogenic differentiation potential to the early adherence subsets. Moreover, late adherent cells exhibited distinct pattern of paracrine secretome including higher level secretion of cytokines than the early adherent subsets. Taken together, these results suggest the possibility that distinct adherence properties of MSCs can be another parameter of clonal heterogeneity in the subpopulations of adipose tissue MSCs and that it can be an important factor for optimization of MSC preparation for cell therapeutic trials.