The identification of human CD34-negative (CD34−) hematopoietic stem cells (HSCs) provides a new concept for the hierarchy in the human HSC compartment. This study investigated the long-term repopulating capacity and redistribution kinetics of human cord blood-derived CD34− severe combined immunodeficiency (SCID)-repopulating cells (SRCs) and compared them with those of CD34+ CD38+ and CD34+ CD38− SRCs using the intra-bone marrow injection (IBMI) to clarify the characteristics of CD34− SRCs. On the basis of the limiting dilution analyses data, estimated numbers of CD34+ CD38+, CD34+ CD38−, and CD34− SRCs were transplanted to NOD/SCID mice by IBMI. The human cell repopulation at the site of injection and the other bones were serially investigated. Interestingly, CD34+ CD38+, CD34+ CD38−, and CD34− SRCs began to migrate to other bones 2 and 5 weeks after the transplantation, respectively. Accordingly, the initiation of migration seemed to differ between the CD34+ and CD34− SRCs. In addition, CD34+ CD38+ SRCs only sustained a short-term repopulation. However, both CD34+ CD38− and CD34− SRCs had longer-term repopulation capacity. Taken together, these findings showed that CD34− SRCs show different in vivo kinetics, thus suggesting that the identified CD34− SRCs are a distinct class of primitive HSCs in comparison to the CD34+ CD38+ and CD34+ CD38− SRCs.