Potential sources of error in surgical pathology include specimen misidentification, unidentified tissue, and tissue contamination of paraffin blocks and slides. Current molecular approaches to characterize unidentified or misidentified tissue include fluorescence in situ hybridization identification of sex chromosomes (XY FISH) and microsatellite analysis. Polymorphic deletion probe (PDP) FISH, a novel FISH assay based on copy number variants, can distinguish between cells and tissues from 2 individuals in situ, independent of gender. Using a panel of 3 PDPs, we compared the genotypes of potential tissue contaminants (n=19) and unidentified tissues (n=6) with patient tissues to determine the utility of PDP FISH in resolving specimen identity. XY FISH was added to increase the informative potential of the assay, and microsatellite analysis was used as a gold standard to confirm PDP FISH results. PDP FISH distinguished between putative contaminants and patient tissues in 13 of 14 cases and indicated a high likelihood of 2 tissues originating from the same source in 11 of 11 cases. The assay has a sensitivity and specificity of 86% [6/7, exact 95% confidence interval (CI): 42%, 97%] and 100% (9/9, exact 1-sided 97.5% CI: 68%, 100%), respectively, and a positive predictive value and negative predictive value of 100% (6/6, exact 1-sided 97.5% CI: 54%, 100%) and 90% (9/10, exact 95% CI: 55%, 98%), respectively. PDP FISH is an accurate and practical molecular assay for the genetic characterization of potential tissue contaminants and unidentified tissues, especially in the setting of small sample size, and permits concomitant assessment of morphology.