e21550 Background: The differentiation between benign melanocytic neoplasms (nevi) and malignant melanocytic neoplasms (melanoma) presents a significant diagnostic challenge, necessitating the development of accurate and reliable diagnostic markers. This study aimed to evaluate the analytical characteristics of selected DNA markers for their efficacy in differentiating these conditions. Methods: A prospective analysis was conducted on sample groups obtained from participants of the multicenter clinical study, MoleMed. The study included 112 histological samples from 100 patients and a corresponding number of cytological samples, collected with the aim of evaluating DNA markers' sensitivity and specificity. DNA extraction followed a previously developed protocol, with concentration and quality assessed through microchip electrophoresis and real-time PCR. Molecular testing targeted mutations in "hotspots" of the BRAF and NRAS genes, as well as promoter mutations in the TERT gene, employing mutation-specific PCR amplification and Sanger sequencing for verification. Results: DNA extraction was successfully performed for 101 cytological samples, with concentrations ranging from < 0.001 ng/µl to 5,508 ng/µl; the average concentration was 106.52 ng/µl, and the median was 2.37 ng/µl. BRAF mutations were found in 28 samples, with the mutation c.1799T > A (p.Val600Glu, V600E) in 21 samples, c.1798_1799GT > AA (p.Val600Lys, V600K) in 6 samples, and c.1799_1800TG > AT (p.Val600Asp, V600D) in 1 sample. NRAS mutations were detected in 11 samples, including c.181C > A (p.Gln61Lys, Q61K) in 6 samples, c.182A > G (p.Gln61Arg, Q61R) in 3 samples, and c.182A > T (p.Gln61Leu, Q61L) in 2 samples. No cases of simultaneous presence of more than one mutation in BRAF and NRAS "hotspots" within the same cytological sample were observed. DNA profiling of cytological material revealed signs of malignant neoplasms (MN) in 49 samples and no signs of MN in 52 samples. TERT mutations c.-124C > T were detected in 21 samples, c.-146C > T in 31 samples (including "borderline" cases), and c.-139_-138CC > TT in 4 samples. Six samples exhibited more than one mutation, highlighting the complexity of genomic alterations in these neoplasms. Conclusions: The PCR analysis of the five DNA markers on both histological and cytological samples of human skin melanomas and nevi demonstrates promising analytical characteristics, with sensitivity and specificity exceeding 70% for all tested scenarios. These results highlight the potential utility of these DNA markers in the differential diagnosis of melanocytic neoplasms, offering a reliable, non-invasive diagnostic tool that complements traditional histopathological examination. Clinical trial information: NCT04353050 .