Objective To isolate ES cell colonies in 615 mouse. Method\ ES cell colonies in 615 mouse were isolated by PMEF feeder layer and the primary examination of these colonies without feeder layer was made. Results\ The formation rate of ES cell colonies and the rate of successful propagation were 22.6% and 0.94%,with positive ALP staining, stable karyotype and differentiation ability. Conclusion\ ES cell colonies in 615 mouse were isolated successfully.