HOBX13 is a transcription factor expressed in the normal prostatic glands and overexpressed in prostate cancer. Recent studies suggested that HOXB13 represents a prostate-specific marker in the differential diagnosis between prostatic and urothelial carcinoma. The aim of this study was to analyze and compare the diagnostic value of HOXB13 and prostate-specific antigen (PSA) immunoexpression for the detection of prostatic origin in metastatic tumours. PSA and HOXB13 immunohistochemical expression was assessed in 50 metastatic tumors, including 15 metastases from prostatic adenocarcinoma, 11 from lung adenocarcinoma, 12 from urinary bladder urothelial carcinoma, 11 from colorectal carcinoma, and in 1 from hepatocellular carcinoma. Strong staining for HOXB13 was observed in >75% of neoplastic cells in 15/15 (100%) metastases from prostate cancer. Weak staining in <25% of cells was found in 2/12 (17%) metastases from urothelial carcinoma. PSA immunostaining was detected only in 8 (53%) cases of prostatic origin. The sensitivity and specificity for metastatic prostate cancer were 100% and 94% for HOXB13 and 53% and 100% for PSA. Due to its high sensitivity and specificity, HOXB13 may be included in the pool of prostate-specific markers in metastases showing absent or weak staining for PSA before excluding prostatic derivation.