Background/Objectives: Melanoma is an aggressive skin malignancy, and the majority of deaths associated with melanoma result from malignant skin lesions. Our study aims to evaluate the expression of the markers CD133 and NANOG, associated with tumor stem cells, and to analyze their link with epidemiological and histological parameters, thus contributing to early diagnosis and the development of targeted therapies. Methods: We performed a retrospective study in the Mureș Clinical County Hospital, Romania, which included 66 cases of melanoma: 50 primary cutaneous melanomas, 10 metastases, and 6 local recurrences. CD133 and NANOG marker expression was assessed by immunohistochemistry and quantified using the H score. Statistical analyses were applied to determine the correlations between marker expression and clinicopathological parameters. Results: CD133 expression was identified in six cases (12%) of primary melanoma, with a mean H-Score of 29, and was associated with an increased Breslow index and a higher number of mitoses. NANOG expression was positive in 30 cases (60%) of primary melanoma, with a median H-Score of 15 and with increased expression observed in cases with pagetoid migration and lesions in situ. In metastases, eight cases (80%) were positive for NANOG and four (40%) for CD133. Local recurrences showed positive expression for NANOG in four cases (66%). Conclusions: The expression of CD133 and NANOG markers highlights the role of tumor stem cells in melanoma progression. Early identification of these markers could improve diagnosis and treatment, including the application of targeted therapies.