Purpose: Patients with head and neck cancer often develop a lung tumor that can be diagnosed as distant metastasis (DM) or second primary tumor (SPT). In this study, we useTP53 mutation analysis for validation of an allelic loss marker panel and a decision algorithm for distinguishing between DM and SPT. Experimental Design: Tumor pairs of 39 patients were analyzed for TP53 mutations, for patterns of allelic loss and immunohistochemical analysis of p53 expression. Results of these three analyses were compared, using mutation analysis as gold standard. Results: Loss of heterozygosity (LOH) analysis indicated DM in 15 and SPT in 23 cases (one inconclusive). TP53 mutation analysis was informative in 15 cases. Based on the p53 mutation status alone, nine tumors were diagnosed as SPT and six as DM. In all 15 cases the LOH analysis was in concordance with the TP53 mutation analysis. Immunostaining for p53 showed promise as a first scan to diagnose lung tumors as SPT but cannot be used to diagnose DM. Conclusion: The TP53 mutation data validate the suitability of the LOH marker panel and decision algorithm for differential diagnosis of DM and SPT in the lung. LOH analysis can theoretically be exploited in almost all cases and is less laborious thanTP53 mutation analysis.