Leukemic stem cells (LSCs) in acute myeloid leukemia (AML) represent a low-frequency subpopulation of leukemia cells that possess stem cell properties distinct from the bulk leukemia cells, including self-renewal capacity and drug resistance. Due to these properties, LSCs are supposed to facilitate the development of relapse. The existence of LSCs is demonstrated by the ability to engraft and initiate human AML in immune-compromised mouse models. Although several lines of evidence suggest the complex heterogeneity of phenotypes displayed by LSC, many studies consider the CD34+/CD38- compartment as the most relevant. To increase the understanding of the true LSC, techniques such as multicolor flow cytometry, side-population assay and ALDH assay are utilized in many laboratories and could aid in this. A better understanding of different LSC phenotypes is necessary to enhance risk group classification, guide clinical decision-making and to identify new therapeutic targets. These efforts to eliminate LSC should ultimately improve the dismal AML outcome by preventing relapse development.