Objective: The aim of this investigation was to explore the expression dynamics of HOXB13, a pivotal transcription factor implicated in prostate gland differentiation, across various phases of prostate cancer development and advancement. Methods: In this study, we undertook the validation of a HOXB13 antibody using genetic controls and proceeded to assess HOXB13 protein expression in both murine and human developing prostates, as well as in localized prostate cancers and metastatic castration resistant prostate cancers. Results: Conclusion: Our findings revealed an escalation in HOXB13 expression during the latter stages of murine prostate development. Notably, all localized prostate cancers exhibited discernible HOXB13 protein expression. Intriguingly, lower levels of HOXB13 expression were discerned in tumors of higher grade, although no significant correlation was established between HOXB13 expression and the likelihood of recurrence or disease-specific survival. Conversely, the majority of advanced metastatic prostate cancers demonstrated sustained HOXB13 expression, encompassing both androgen receptor-negative castration-resistant prostate cancers and neuroendocrine prostate cancers. Furthermore, reduced HOXB13 expression in neuroendocrine prostate cancers was linked to HOXB13 gene body CpG methylation. When compared to the widely employed prostate lineage marker NKX3.1, HOXB13 exhibited superior sensitivity in detecting advanced metastatic prostate cancers. Additionally, immunohistochemistry for HOXB13 exhibited robust sensitivity and specificity for confirming the prostatic origin in a patient cohort comprising both prostatic and non-prostatic tumors. These study outcomes furnish valuable insights into the expression profile of HOXB13 throughout prostate development and the trajectory of cancer progression. HOXB13 emerges as a promising diagnostic biomarker for prostate cancer, particularly in affirming the prostatic origin of advanced metastatic castration-resistant tumors.