Background: The incidence of transformation to neuroendocrine prostate cancer (NEPC) has increased in castration resistant prostate cancer (CRPC) in parallel with treatment advances inhibiting androgen receptor signaling. The current understanding is that this occurs in ̃10-20% of CRPC cases. Missing is a determination of the timing of molecular events that drive the process. Methods: Under an IRB-approved protocol, retrospective annotation of all MSK-reviewed pathology reports was conducted for 1447 prostate cancer patients with MSK-IMPACT sequencing data. For patients with pathologically confirmed NEPC, the date of the first sample with unequivocally reported NEPC (described as “neuroendocrine carcinoma” or as having “neuroendocrine differentiation” or “neuroendocrine features”) was recorded. Patients with early signs of histologic transformation not specifically reported as NEPC (double negative prostate cancer or rare/focal staining for NE markers) were analyzed separately. Sequencing results were described by castration-status at collection (CRPC vs castration-sensitive) and, if applicable, the relationship to NEPC diagnosis (i.e. pre- vs post-NEPC). Genomic enrichment analysis was used to identify differentially altered genes between groups. Results: In total, 95 (6.6%) patients had pathologically confirmed NEPC during their disease course, from whom 150 samples with sequencing results were available: including 18 patients with matched pre- and post-NEPC samples. CRPC samples from patients with NEPC (n = 70) were significantly enriched for RB1 alterations (50% vs 12%, p < 10^-10, q < 10^-10). AR alterations were significantly enriched in CRPC samples from patients without NEPC (n = 380) (63% vs 21%, p < 10^-10, q < 1.27*10^-8). Further, alterations in numerous genes including TP53, AMER1, ARID5B, YAP1, SOX2, and NKX2.1 were enriched in NEPC patients at the 95% confidence interval (CI) without correction for repeat testing. Matched pre- and post-NEPC samples demonstrated that TP53 alterations in post-NEPC samples are detected in the majority of pre-NEPC samples (8 of 10 patients), but RB1 alterations in post-NEPC samples are detected in a minority of pre-NEPC samples (1 of 8 patients). 54 (3.7%) patients had evidence of early histologic transformation. CRPC samples from these patients (n = 29) were enriched for mutations in RB1, MAP2K2, MUTYH, and CTNNB1 at the 95% CI without correction. FOXA1 mutations were enriched in patients without transformation. Conclusions:RB1, consistent with previous findings, is enriched in NEPC. The inability to detect RB1 alterations in pre-NEPC samples supports divergent evolution, although technical limits of tissue panel sequencing make it difficult to rule out the presence of sub-clonal alterations. Further study of additional genes which contribute to histologic transformation and the development of NEPC is warranted.