Mesenchymal stem cells (MSCs) derived from human amnion have both self-renewal capability and multipotency and are an attractive cell source for cell-based therapy. However, these cells have been shown to be heterogeneous, and as of yet no single-cell-derived MSCs clone has been established from human amnion. This study was carried out to isolate MSCs clones by limiting dilution method and compare their characteristics in vitro. Three clones (namely, 8B, 11D, and 11F) were established from a heterogeneous population of human amnion-derived cells (h-hAMCs). The clones and h-hAMCs successfully proliferated while demonstrating different cumulative population doublings (CPD) during an 80-day culture. In addition, the colony-forming efficiency (CFE) of h-hAMCs was significantly lower than those of 8B and 11F and higher than that of 11D. Clones 8B and 11F were tripotent, whereas 11D did not undergo chondrogenic differentiation. All cells expressed surface markers including CD29, CD44, and CD105 and notably, the clones expressed higher levels of CD105 than h-hAMCs (95.96, 97.05, 98.14% and 72.81% for 8B, 11D, 11F and h-hAMCs, respectively). In addition, the expression of stem cell gene Nanog-3 was associated with the differential differentiation potential of 11D from 8B, 11F, and h-hAMCs. These results suggested that significant differences existed between individual hAMCs. Further studies for developing novel methods to select sub-populations of hAMSCs are warranted for their clinical applications, in which CD105 and stem cell gene Nanog-3 are possible candidate markers.