Purpose <p>Prostate-specific membrane antigen (PSMA) PET/CT enhances prostate cancer (PCa) diagnosis. The newly developed PSMA probe, [⁶⁸Ga]Ga-P16-093, with low urinary excretion, has shown superior diagnostic efficacy compared to conventional PSMA probes. This study aims to assess the diagnostic efficacy and local–regional staging performance of [⁶⁸Ga]Ga-P16-093 in PCa lesions of newly diagnosed patients, using histopathology as the gold standard for validation.</p> Methods <p>This prospective study enrolled newly diagnosed PCa patients (April 2022–November 2023). All patients underwent [⁶⁸Ga]Ga-P16-093 PET imaging, and a subset also received [⁶⁸Ga]Ga-PSMA-11 PET/CT within one week. Radical prostatectomy within two weeks post-PET provided complete pathological specimens from all patients. The diagnostic efficacy and locoregional staging performance of [⁶⁸Ga]Ga-P16-093 were statistically compared with that of [⁶⁸Ga]Ga-PSMA-11, using histopathology as the gold standard.</p> Results <p>Fifty-six treatment-naïve male patients (mean age 67 ± 6&#xa0;years; range 52–77) were prospectively enrolled. A subgroup of 37 patients who underwent both [⁶⁸Ga]Ga-P16-093 and [⁶⁸Ga]Ga-PSMA-11 PET/CT was analyzed for direct comparison. [⁶⁸Ga]Ga-P16-093 PET/CT demonstrated superior diagnostic performance in primary prostate cancer evaluation, with sensitivity of 74.44% (201/270, 95% CI: 68.38–80.50%), specificity of 96.26% (387/402, 95% CI: 94.39–98.15%), and accuracy of 87.50% (588/672, 95% CI: 85.05–89.95%). Compared with [⁶⁸Ga]Ga-PSMA-11 PET/CT, [⁶⁸Ga]Ga-P16-093 PET/CT showed higher tracer uptake, with SUV_max of 9.86 ± 6.82 vs. 6.74 ± 1.89 (<i>P</i> = 0.041), SUV_mean of 5.81 ± 4.03 vs. 3.98 ± 1.04 (<i>P</i> = 0.037), and T/B ratio of 23.19 ± 17.51 vs. 16.04 ± 7.75 (<i>P</i> = 0.042). Additionally, ROC analysis revealed a significantly greater AUC for P16-093 (0.85 vs. 0.75, <i>P</i> &lt; 0.05). Moreover, locoregional staging accuracy was higher at 59.46% (22/37) compared with 32.43% (12/37) for [⁶⁸Ga]Ga-PSMA-11 PET/CT.</p> Conclusion <p>[⁶⁸Ga]Ga-P16-093 PET/CT exhibits high diagnostic performance in the diagnosis of primary PCa and shows significant advantages in identifying local tumor segments. [⁶⁸Ga]Ga-P16-093 may serve as an alternative to [⁶⁸Ga]Ga-PSMA-11 in the future diagnosis of PCa.</p> Trial registration <p>ClinicalTrials.gov, NCT05324332. Registered 04 March 2022.</p> URL of registry <p><a href="https://clinicaltrials.gov/ct2/show/NCT05324332">https://clinicaltrials.gov/ct2/show/NCT05324332</a></p>