Objective The aim of this study was to explore the possibility to establish single-cell colonal lines from chicken embryonic primordial germ cells(EPGCs) and investigate their biological characteristics.Method single-cells were derived from the 4th passages and multiplied clonal EPGCs at stage 19 and 28,which were digested with 0.25% trypsin-EDTA.Then the single cell was picked up and seeded into individual well of a 96-well plate containing fibroblast feeder-layer,and every well contained one cell.The outgrowth of single-cell clones were passed after treatment with trypsin-EDTA,and their biological characteristics were detected by cytochemistry and histoimmunochemistry to detect their special markers.The karyotypes were assessed by routine test.The pluripotency was analyzed by differentiation in vitro.Results Nine in total of 288 clones maintained the normal diploid karyotype and undifferentiated.7 clones were passaged for 2 passages,and 2 clones were passaged for 4 passages.The 9 clones possessed normal karyotypes,expressing a series of surface markers such as: alkaline phosphatase and stage-specific embryonic antigen(SSEA)-1.Conclusion Nine single cell-cloned of chicken embryonic PGCs have been derived in our laboratory.The cells possess stable biological characteristics of undifferentiated PGCs.