Background Melanoma skin cancer remains the leading cause of skin cancer–related deaths. Spitz lesions represent a subset of melanocytic skin lesions characterized by epithelioid or spindled melanocytes organized in nests. These lesions occupy a spectrum ranging from benign Spitz and atypical Spitz lesions all the way to malignant Spitz tumors. Appropriate management is reliant on accurate diagnostic classification, yet this effort remains challenging using current light microscopic techniques. The discovery of novel biomarkers such as microRNAs (miR) may ultimately be a useful diagnostic adjunct for the evaluation of Spitz lesions. miR expression profiles have been suggested for non-Spitz melanomas but have yet to be ascribed to Spitz lesions. We hypothesized that distinct miR expression profiles would be associated with different lesions along the Spitz spectrum. Materials and methods RNAs extracted from paraffin-embedded, formalin-fixed tissues of 11 resected skin lesions including benign nevi (n = 2), benign Spitz lesions (n = 3), atypical Spitz lesions (n = 3), and malignant Spitz tumors (n = 3) were analyzed by the NanoString platform for simultaneous evaluation of over 800 miRs in each patient sample. Results Benign Spitz lesions had increased expression of miR-21-5p and miR-363-3p compared with those of benign nevi. Malignant Spitz lesions exhibited overexpression of miR-21-5p, miR-155-5p, and miR-1283 relative to both benign nevi and benign Spitz tumors. Notably, atypical Spitz tumors had increased expression of miR-451a and decreased expression of miR-155-5p expression relative to malignant Spitz lesions. Conversely, atypical Spitz lesions had increased expression of miR-21-5p, miR-34a-5p, miR-451a, miR-1283, and miR-1260a relative to benign Spitz tumors. Conclusions Overall, distinct miR profiles are suggested among Spitz lesions of varying malignant potential with some similarities to non-Spitz melanoma tumors. This work demonstrates the feasibility of this analytic method and forms the basis for further validation studies.