The study of melanocyte biology and pathology is currently limited by an inability to prospectively isolate distinct melanocytic subtypes, such as melanocyte progenitors and their pigment-producing differentiated progeny, from human and mouse skin. As a result, current efforts in this field are restricted to lineage tracing and cell isolation from genetically engineered mice expressing fluorescent markers, which depend on the fidelity of Cre recombinase promotion, and the use of selective media to promote melanocyte clonogenicity in vitro, which may be artefactual. Here we describe the isolation within c-Kit-positive melanocytes from human and mouse tail epidermis of phenotypically distinct melanocyte subpopulations associated with the production of melanin, the canonical marker of melanocytic differentiation. Exploiting the autofluorescence of melanin pigment upon excitation with near-infrared (IR) light, in combination with the increased side-scatter seen in pigmented cells, we identified putative non/low-pigmented melanocyte progenitors and more differentiated pigmented melanocytes, whose clonogenicity and phenotypes were subsequently evaluated. This advance will facilitate the study of normal and neoplastic melanocyte development.