Energy metabolism is the foundation of survival for all organisms, and mitochondria are the most important energy‐supplying organelles in eukaryotic cells. However, the mitochondrial and energy/metabolism‐related properties of cancer stem cells (CSCs), the stem cell‐like subpopulation in tumor masses, remain unknown. In our study, we compared the masses of mitochondria and mitochondrial DNA (mtDNA), the mitochondrial membrane potential (Δψm), oxygen/glucose consumption, and the concentration of reactive oxygen species (ROS) and ATP between lung CSCs (LCSCs) and non‐LCSCs. In addition, the change in features during differentiation was examined. Some mitochondrial and energy metabolism‐related properties, such as perinuclear mitochondrial distribution, a lower quantity of mtDNA, higher Δψm, lower oxygen/glucose consumption, and lower intracellular concentrations of ROS and ATP, can be used as indicators of LCSCs.