Background: The somatic mutation HSD3B1(1245A > C) has been mechanistically linked to castration-resistant prostate cancer by encoding a mutant enzyme that augments intratumoral dihydrotestosterone (DHT) synthesis. Given the HSD3B1(1245C) allele is also frequently found in the germline, we hypothesized men inheriting this variant allele would exhibit resistance to androgen deprivation therapy (ADT). Methods: We determined HSD3B1 genotype retrospectively in men treated with ADT for post-prostatectomy biochemical failure. We analyzed progression-free survival (PFS), distant metastasis-free survival (DMFS), and overall survival (OS) according to HSD3B1 genotype using Kaplan-Meier methods. Demographic and treatment characteristics were compared to assess for potential confounders using Fisher’s exact test and Kruskal-Wallis analysis of variance. Multivariable analyses were performed to assess the independent predictive value of HSD3B1 genotype on outcomes. Results were independently validated in two external cohorts, including a second post-prostatectomy cohort and a metastatic cohort. Results: 443 patients were included: 118 in the primary cohort, 137 in the post-prostatectomy validation cohort, and 188 in the metastatic validation cohort. In the primary cohort, median PFS diminished as a function of the number of variant alleles inherited (6.6 years in homozygous wild-type men, 4.1 years in heterozygotes, and 2.5 years in homozygous variants; P = 0.01). Median DMFS likewise diminished according to the number of variant alleles inherited (9.1 vs. 6.8 vs. 3.6 years, respectively; P = 0.01). Finally, OS decreased with the number of variant alleles inherited (5-year and 10-year OS: 82% and 55% vs. 74% and 35% vs. 58% and 0%; P = 0.006). On multivariable analysis, the impact of HSD3B1 genotype on metastasis (hazard ratio (HR) 2.8; P = 0.023) and death (HR 3.3; P = 0.016) was maintained. Findings in the external cohorts independently validated the profound impact of HSD3B1(1245C) on outcomes, including overall survival. Conclusions: Inheritance of the HSD3B1(1245C) allele that enhances dihydrotestosterone synthesis predicts innate resistance to ADT in prostate cancer.