Background: TMB is an emerging biomarker to predict clinical benefit from immune checkpoint inhibitors (ICI). Although high TMB in prostate cancer is rare, and we sought to evaluate independent associations of TMB by clinical factors, prior treatment, and genomics. Methods: This study used the US-based nationwide de-identified Flatiron Health-Foundation Medicine prostate cancer clinico-genomic database. The de-identified data originated from approximately 280 US cancer clinics (~800 sites) between January 2011-March 2022. Multivariable linear regression model was used to assess independent prediction of TMB levels, which were log2+1 transformed to better normalize distribution. Data are expressed as (estimate, [95% CI], p-value). Results: The study included 2,748 tissue specimens: 51.7% were from the prostate, 10.3% from bone, and 14.6% from lymph nodes. Specimens from white patients comprised 61.2% of samples, with another 7.5% from Black, 1.1% from Asian, and 30% from other or unknown race patients. Of our cohort, 58.5%, 15.5%, and 26.0% were from patients who received 0, 1-24 months, and >24 months of androgen deprivation therapy (ADT), respectively. Also 19.7%, 1.8%, 11.4%, and 0.4% had any exposure to novel hormonal therapies (NHT), Radium-223, taxanes, and ICI, respectively. Higher TMB was independently associated with ADT use >24 months (0.16, [0.05-0.27], p=0.005) and Asian race (0.36, [0.03-0.69], p=0.034). Prior treatment with NHT, taxanes, or radium was not associated with higher TMB. Compared to prostate, bladder (0.22, [0.03-0.41], p=0.023), liver (0.27, [0.13-0.42], p<0.001), and other (0.29, [0.15-0.43], p<0.001) biopsy sites were associated with high TMB, but lymph node biopsy was not. MSI-high (2.30, [1.96-2.64], p<0.001), MSI-intermediate (0.51, [0.14-0.88], p=0.007), and MSI-unknown (0.84, [0.60-1.09], p<0.001) were associated with increased TMB. Individually altered genes associated with increased TMB included MLH1 (0.77, [0.31-1.24], p=0.001), MSH2 (1.09, [0.74-1.45], p<0.001), MSH3 (0.64, [0.018-1.11], p=0.006), MSH6 (0.94, [0.61-1.26], p<0.001), BRCA2 (0.56, [0.43-0.69], p<0.001), CDK12 (0.38, [0.24-0.53], p<0.001), FANCA (0.42, [0.07-0.77], p=0.02), MRE11 (0.87, [0.11-1.62], p=0.025), and PALB2 (0.83, [0.48-1.18], p<0.001). No individual gene was independently associated with lower TMB. Conclusions: TMB associates with MSI status and MMR genes. However, aside from associating with long term ADT use, TMB does not have strong association with prior systemic treatment. Certain biopsy specimen sites including bladder and liver have increased TMB but overall, the association is relatively weak in comparison to MMR genes and MSI status. This study allows better understanding of how prior treatments, clinical and genomic factors affect TMB status, which may add value to MSI status in predicting treatment response in advanced prostate cancer.