Gene expression-based assays have been introduced into the clinical arena to assist in the diagnosis of poorly differentiated or undifferentiated tumors. The US Food and Drug Administration has cleared the microarray-based Pathwork Tissue of Origin (TOO) Test (Pathwork Diagnostics, Sunnyvale, CA) for the molecular characterization of such challenging specimens. We aimed at verifying the analytic and clinical performance of this test on 43 poorly differentiated and undifferentiated tumor samples, including 6 off-panel cases and 7 cancers of unknown primary (CUP). Our results showed 97% (95% confidence interval, 80.4%-99.8%) agreement between the Pathwork TOO Test result and the complete diagnosis, which included clinical correlations and immunohistochemical staining, after the original diagnosis. We concluded that for off-panel and CUP samples, the tissue type and the cell type may be confounded by the Pathwork TOO Test and that careful clinicopathologic assessment is needed when interpreting results from this helpful ancillary tool for pathologists.