MicroRNAs are small non-coding RNAs, which inhibit expression of specific target genes at the post-transcriptional level and are often misregulated in human cancer. Among them, miR-34a is considered a tumor suppressor with a hypothetical role in melanoma tumorigenesis. In this work, 62 Italian index patients with familial melanoma and negative for CDKN2A/CDK4 screening were investigated for miR-34a germline mutations. Eight novel miR-34a sequence variants were identified at both the heterozygous (c.+259G>A, c.+424G>A, c.+1465C>T, c.+1769C>T, c.+2456T>G, c.+2603C>T, c.+2972T>A, c.+3069T>C) and homozygous (c.+424G>A, c.+1465C>T, c.+1769C>T) states. Molecular screening identified all nucleotide changes in a healthy population of 150 controls and demonstrated that they are common polymorphisms. However, statistically significant differences of allele and genotype frequencies were detected for c.+1465C>T and c.+1769C>T, and borderline values for c.+2456T>G. By stratifying patients by relevant clinical features (presence/absence of multiple primary melanoma, Breslow's thickness, phototype and number of nevi), no significant findings were noted except for an association between the c.+424G>A (heterozygous individual GA) and multiple primary melanoma and phototype III-IV. Our preliminary study suggests that miR-34a, although having a role in late tumorigenesis, does not contribute to the inherited susceptibility to cutaneous melanoma. A function as phenotypic modulator in familial melanoma cannot be excluded.