CD34 and CD133 are the most commonly used markers to enrich hematopoietic stem cells (HSCs). Positively selected HSCs are increasingly used for autologous and allogeneic transplantation, yet the biological properties of CD34(+) and CD133(+) cells are largely unknown. In the present study, a genome-wide gene expression analysis of human cord blood (CB)-derived CD34(+) cells was performed. The CD34(+) gene expression profile was compared to an identically constructed CD133(+) gene expression profile to reveal the specific expression patterns and major differences of CD34(+) and CD133(+) cells. As expected, many genes were similarly expressed in the two cell populations, but cell-type-specific gene expression was also demonstrated. Self-organizing map analysis was used to identify transcripts having similar expression patterns, and the results were compared between CD34(+) and CD133(+) cells. Also, a prioritization algorithm was used to rank the genes best separating CD34(+) and CD133(+) cells from their CD34() and CD133() counterparts in CB. Our results show that CD133(+) cells have higher numbers of up-regulated genes than CD34(+) cells. Furthermore, the uniquely expressed genes in CD34(+) or CD133(+) cell populations were associated with different biological processes. CD34(+) cells overexpressed many transcripts associated with development and response to stress or external stimuli. In CD133(+) cells, the most significantly represented biological processes were establishment and maintenance of chromatin architecture, DNA metabolism, and cell cycle. The differences between the gene expression profiles of CD34(+) and CD133(+) cells indicate the more primitive nature of CD133(+) cells. These profiles suggest that CD34(+) and CD133(+) cells may have different roles in hematopoietic regeneration.