Background: MTP may predict the tissue of origin in CUP and thus improve therapy for some patients. The ability of MTP to improve diagnosis over standard pathology is undefined. We evaluated MTP on biopsies to assess accuracy and diagnostic ability. Methods: From March 2008–January 2010 clinicopathologic data from 171 CUP patients who had MTP performed (bioTheranostics) on paraffin embedded biopsies were collected. The MTP diagnoses were validated by correlation with: 1) latent primaries found months later; 2) initial diagnosis by immunohistochemistry (IHC); 3) additional IHC and/or clinical findings after MTP; 4) clinical features, and 5) outcome of colorectal MTP patients treated with site-specific chemotherapy. Results: MTP provided a diagnosis in 144 of 171 tumors (84%); 22 had insufficient tumor for MTP (13%) and 5 were unclassifiable (3%). Histopathologic diagnoses were adenocarcinoma 56%, poorly differentiated carcinoma 27%, and others 17%. MTP diagnosed 21 tumor types [colorectal (16%), lung (11%), breast (9%), liver (6%), pancreas (5%), ovary (5%), other (48%)]. 18 of 24 patients with latent primaries had a correct diagnosis by MTP (75%). A specific IHC diagnosis was made in 52 patients (30%) and in 40 (77%) MTP diagnosis agreed (100% concordance breast, 93% colorectal and 74% lung). An IHC diagnosis of 2 or 3 possible types was made in 97 patients (70%) and in 43 MTP diagnosis agreed with 1 IHC diagnosis (44%). In 54 patients MTP disagreed with any IHC diagnosis (56%). In 41 (75%) the clinical features were consistent with MTP diagnosis and in 18 of 25 (72%) additional IHC and/or clinical findings supported MTP diagnosis. Twenty-one patients with colorectal MTP diagnosis received colorectal chemotherapy (response rate 70%, median survival 21 months). Conclusions: MTP predicts a tumor type in most CUP patients and the accuracy was supported by a high level of agreement with: identified latent primaries (75%); specific IHC diagnoses (77%); additional directed IHC and/or clinical findings (72%); clinical features (75%); expected outcome in the colorectal subset treated with colorectal chemotherapy. MTP is a valuable test in CUP and complements standard diagnostic pathology.