Objective To investigate the efficiency of isolation and cloning of ES cell colonies in Kunming mouse. Method\ Three culture conditions were adopted,namely, PMEF feeder layer, NIH3T3 feeder layer and LIF without feeder layer. Results\ According to the formation rate of ES cell colonies, the two kinds of feeder layer are better than LIF only, the blastocysts are better origin for ES cells than morulae and there are no differences between the two kinds of feeder layer. Conclusion It is a more efficient way to isolate and clone ES cell colonies from Kunming mouse blastocysts by PMEF or NIH3T3 feeder layer.