BACKGROUND: To standardize the separation method of cord blood and establish the Korean Cord Blood Bank, we analysed the data regarding cord blood separation from 8 different hospitals which involved in cord blood banking project. METHODS: We analysed 306 samples which collected with heparinized syringes and separated within 24 hours by Ficoll-Hypaque density gradient separation method and RBC depletion method with 3% gelatin. We performed the mononuclear cell (MNC) count, viability, CD34+ cell count and CFU-GM count following separation of cord blood. We assessed the yield of hematopoietic stem cells by different separation methods in 9 hospitals and the variability among hospitals which performed cord blood separation by same method. RESULTS: 1) In 204 samples of cord blood separated with Ficoll-Hypaque, there were significant differences in MNC count, viability, CD34+ cell count and CFU-GM count among all engaged hospitals, but not in each individual hospital. 2) In 102 samples of cord blood separated with 3% gelatin, there were significant differences in viability, CFU-GM count and CD34+ cell count among all engaged hospitals, but not in MNC count. 3) The yield of MNCs, CD34+ cells and CFU-GM was higher in the cord blood separated with 3% gelatin than Ficoll-Hypaque. CONCLUSION: We suggest that RBC depletion method with 3% gelatin would be better than Ficoll-Hypaque method in order to increase the yield of hematopoietic stem cells and standardize the method of cord blood separation.