Non-neoplastic epithelial lesions of the vulva (NNEDV) lichen sclerosus (LS) and squamous hyperplasia (SH) have been implicated in the pathogenesis of squamous cell carcinoma of the vulva (SCC). To date, there have been no recognisable precursor lesions for SCC associated with NNEDV. TP53 is the most frequent genetic change in human cancers and can indicate both aetiology and molecular pathogenesis of tumours. A total of 27 SCC patients underwent immunohistochemistry (IHC) and TP53 mutational analysis using microdissection and direct sequencing. There were 19 patients with areas of adjacent epidermis: 17 had NNEDV (four SCCs had more than one adjacent lesion) and two had normal epidermis. In all, 70.4% of the SCCs, 40% LS and 22.2% SH demonstrated overexpression of p53. In total, 77.8% of SCCs, 46.7% of LS and 22.2% SH demonstrated mutations in TP53, with the majority of lesions having a mutation in codon 136. Eight cases were identified where the same mutation was identified in the SCC and in the adjacent area. These data suggest that TP53 mutations develop in NNEDV and are intrinsic to the clonal evolution that leads to SCC. The type of mutation detected is more likely to occur due to endogenous cellular changes rather than exogenous carcinogen exposure.