Skeletal muscles have the ability to regenerate after injury thanks to the presence of muscle stem cells, called satellite cells (SC). These SC express the Pax7 transcription factor, are essential for muscle regeneration and represent a potential cellular source in the treatment of patients with muscular diseases. However, their in vitro amplification, necessary to obtain a sufficient number of cells before injection, leads to a significant reduction in their regenerative capacity. An alternative may be the use of myogenic stem cells generated in vitro, called reserve cells (RC), which have many similarities to SC. In this study, we wanted to better characterize human RC, at the cellular and molecular level. We have observed that human RC are a heterogeneous population of myogenic stem cells with different levels of Pax7 expression, Pax7High RC and Pax7Low RC with the Pax7High RC that can represent an interesting population for transplantation. For this reason, we performed a sorting to analyse the molecular differences between Pax7High RC and Pax7Low RC. Pax7High RC showed an increase in the expression of Pax3, Pax7, Hes1 and EGFR genes compared to the Pax7Low RC. At the cellular level, we have demonstrated heterogeneity in the expression of Pax3 in RC, with a positive and a negative population. RC also showed heterogeneity in the expression of surface marker such as ErbB3, in their autofluorescence level and in their frequency of cell division.