Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency level of stem cells. A mammalian conserved cluster of miRNAs encoded by this region exhibited significant expression differences between full- and partial-pluripotent stem cells. Several miRNAs from this cluster potentially target the PRC2 silencing complex, and may form a feed-forward regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full-pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels, therefore the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or ES cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells, and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy. The putative synergetic effects of several miRNAs also furthered the understanding of the coordinative functions of miRNAs in regulating complex biological processes.