Abstract: Spermatogonial stem cells (SSCs) were isolated from chicken embryo testis on 16^th hatching days. Growth of SSCs which were cultured in vitro in three culture solutions with or without feeder layer were compared， and the characteristic of SSCs was identified. The results showed that, without feeder layer of fibroblast cells， survival times of SSCs in DMEM， TCM199 and RPMI1640 were 6.5， 6.0 and 3.5 d, respectively， and there were extremely significant differences among in DMEM， TCM199 and in RPMI1640 (P<0.01). With feeder layer of fibroblast cells， survival times of SSCs in DMEM， TCM199 and RPMI1640 were 45.5， 38.0 and 14.0 d, respectively， and there were extremely significant differences among in three culture solutions (P<0.01). Survival time and rate of SSCs cloning in culture system Ⅳ were （45.5±3.2）d and 0.31±0.46, respectively， and those in culture system Ⅳ were extremely significant higher than those in other five cultures(P<0.01). SSCs were subcultured in the culture system Ⅳ to 3rd passage， and rates of cloning of systems at passage 1， 2 and 3 were 31.6%， 20% and 18%, respectively. SSCs formed cell clonies and proliferated， and cell clonies of SSCs were positive to alkaline phosphatase (AKP) and stage specific embryonic antigen-1 (SSEA-1). The 3^rd passage SSCs that had been cultured in vitro not only maintained the undifferentiated state and normal diploid karyotype (2n=78)， but also kept the necessary characteristics of SSCs， which was subcultured in culture system Ⅳ with DMEM and feeder layer of fibroblast cells.