DSS1 and p53 are required for homologous recombination, but, although p53 inactivation has a key function in skin tumorigenesis, there is no clear evidence supporting a function of DSS1. We screened the entire DSS1 coding sequence and p53 exons 5-8 in a series of 60 cases of skin squamous cell carcinoma (SCC). Mutational analysis of p53 revealed tumor-associated mutations in 28 (46.7%) of the cases. No tumor-associated DSS1 mutations were detected; however, the germline DSS1 c.143G>A synonymous polymorphism had a significantly higher frequency in patients with SCC (16.67%) versus healthy subjects (5%). DSS1 expression was evaluated by quantitative real-time RT-PCR and immunohistochemistry. With respect to c.143G>G genotype, SCCs and adjacent normal tissues carrying the c.143G>A polymorphism showed significantly lower DSS1 RNA and protein levels and a prevalent cytoplasmic rather than nuclear localization of DSS1 protein. The assay for mRNA stability revealed that the c.143G>A polymorphism affects DSS1 expression efficiency, but not mRNA decay. We clearly showed that the c.143G>A variant is associated with reduced DSS1 expression at both the RNA and protein levels and with altered traffic of the DSS1 protein from the cytoplasm to the nucleus. These alterations could impair DSS1 function in DNA repair and may be implicated in skin cancer.