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Skin is an easily accessible tissue and a rich source of Schwann cells (SCs). Toward potential clinical application of autologous SC therapies, we aim to improve the reliability and specificity of our protocol to obtain SCs from small skin samples. As well, to explore potential functional distinctions between skin‐derived SCs (Sk‐SCs) and nerve‐derived SCs (N‐SCs), we used single‐cell RNA‐sequencing and a series of in vitro and in vivo assays. Our results showed that Sk‐SCs expressed typical SC markers. Single‐cell sequencing of Sk‐ and N‐SCs revealed an overwhelming overlap in gene expression with the exception of HLA genes which were preferentially up‐regulated in Sk‐SCs. In vitro, both cell types exhibited similar levels of proliferation, migration, uptake of myelin debris and readily associated with neurites when co‐cultured with human iPSC‐induced motor neurons. Both exhibited ensheathment of …