Despite the wide application of ES cell technology, little is known about the pluripotent nature of ES cells. This is partly due to the heterogeneity of ES cell population in culture. This report described the specific labelling of undifferentiated cells in ES cell lines. oct-4 gene is specifically expressed in all the totipotent cells in mouse embryos and undifferentiated ES cells. A constructed pG18NG was obtained by inserting the reporter gene beta geo into the oct-4 transcription elements. ES cell lines MESPU22 and MESPU13 were transfected with this construct and stable integrated cell clones were selected out. With the experiments of in vitro cultivation, differentiation and chimeras production, it was confirmed that we have successfully labelled the undifferentiated cells in ES cell lines, and this label was both valid in vitro and in vivo.