Congenital melanocytic naevi (CMN) can be associated with neurological abnormalities and increased risk of melanoma. Mutations in NRAS, BRAF and Tp53 have been described in individual CMN samples, however, their role in the pathogenesis of multiple CMN within the same subject and development of associated features has not been clear. We hypothesised that a single post-zygotic mutation in NRAS could be responsible for multiple CMN in the same individual, as well as for melanocytic and non-melanocytic central nervous system (CNS) lesions. Fifty-five samples from 15 patients with multiple CMN were sequenced after site-directed mutagenesis and enzymatic digestion of the wild-type allele. Oncogenic missense mutations in codon 61 of NRAS were found in affected neurological and cutaneous tissues of 12/15 patients, but absent from unaffected tissues and blood, consistent with NRAS mutation mosaicism. In ten patients the mutation was consistently c.181C>A, p.Q61K, and in two c.182A>G, p.Q61R. All 11 non-melanocytic and melanocytic CNS samples from five patients were mutation positive, despite NRAS rarely reported as mutated in CNS tumours. Loss of heterozygosity was associated with onset of melanoma in two cases, implying a multi-step progression to malignancy. These results suggest that single post-zygotic NRAS mutations are responsible for multiple CMN and associated neurological lesions in the majority of cases.Journal of Investigative Dermatology ccepted article preview online, 7 February 2013; doi:10.1038/jid.2013.70.