Background: The inability to definitively determine the lineage of neoplasms is less common with modern immunohistochemistry (IHC) and genetic profiling. Nonetheless some PDN defy lineage classification by extensive standard pathologic evaluation. The advent of MTP may provide a new method of improving the diagnosis of these challenging cancers. Methods: A total of 30 of 751 (4%) patients (pts) seen from 2000 – 2012 with cancer of unknown primary (CUP) had PDN without a definitive lineage determined by IHC (median 18 IHC stains, range 9 – 51). From 2008 – 2012 the 30 biopsies had MTP (RT-PCR mRNA CancerTYPE ID, bioTheranostics, Inc.). Additional IHC, genetic sequencing, fluorescent in situ hybridization for specific chromosomal changes and repeat biopsies were performed when feasible to support the MTP diagnosis, and clinical features correlated. Results: MTP lineage diagnoses were made in 25 of 30 (83%), including 10 carcinomas (3 germ cell, 2 neuroendocrine, 5 others), 5 melanomas, 8 sarcomas (3 peritoneal mesothelioma, 1 PNET) and 2 hematopoietic neoplasms (1 lymphoma, 1 chloroma). Additional IHC, genetic testing [BRAF, i(12)p] or repeat biopsies confirmed the MTP diagnoses in 11 of 15 tumors, and the clinical features were consistent with the MTP diagnoses in the majority of patients. Conclusions: This MTP assay can frequently provide a diagnosis for CUP pts and PDN without a definitive lineage defined by extensive IHC. The earlier application of MTP will likely provide an expedited diagnosis, and for some neoplasms is the only test capable of defining lineage and a more specific diagnosis. Appropriate therapy, particularly for pts with germ cell tumors, melanoma, and lymphoma depends on a specific tissue of origin diagnosis.