Objectives. In patients with prostate cancer (PC) receiving prostate-specific membrane antigen- (PSMA-) targeted radioligand therapy (RLT), higher baseline standardized uptake values (SUVs) are linked to improved outcome. Thus, readers deciding on RLT must have certainty on the repeatability of PSMA uptake metrics. As such, we aimed to evaluate the test-retest repeatability of lesion uptake in a large cohort of patients imaged with 18F-DCFPyL. Methods. In this prospective, IRB-approved trial (NCT03793543), 21 patients with history of histologically proven PC underwent two 18F-DCFPyL PET/CTs within 7 days (mean 3.7, range 1 to 7 days). Lesions in the bone, lymph nodes (LN), and other organs were manually segmented on both scans, and uptake parameters were assessed (maximum (SUVmax) and mean (SUVmean) SUVs), PSMA-tumor volume (PSMA-TV), and total lesion PSMA (TL-PSMA, defined as PSMA − TV × SU V mean )). Repeatability was determined using Pearson’s correlations, within-subject coefficient of variation (wCOV), and Bland-Altman analysis. Results. In total, 230 pairs of lesions (177 bone, 38 LN, and 15 other) were delineated, demonstrating a wide range of SUVmax (1.5–80.5) and SUVmean (1.4–24.8). Including all sites of suspected disease, SUVs had a strong interscan correlation ( R 2 ≥ 0.99 ), with high repeatability for SUVmean and SUVmax (wCOV, 7.3% and 12.1%, respectively). High SUVs showed significantly improved wCOV relative to lower SUVs ( P < 0.0001 ), indicating that high SUVs are more repeatable, relative to the magnitude of the underlying SUV. Repeatability for PSMA-TV and TL-PSMA, however, was low ( wCOV ≥ 23.5 %). Across all metrics for LN and bone lesions, interscan correlation was again strong ( R 2 ≥ 0.98 ). Moreover, LN-based SUVmean also achieved the best wCOV (3.8%), which was significantly reduced when compared to osseous lesions (7.8%, P < 0.0001 ). This was also noted for SUVmax (wCOV, LN 8.8% vs. bone 12.0%, P < 0.03 ). On a compartment-based level, wCOVs for volumetric features were ≥22.8%, demonstrating no significant differences between LN and bone lesions (PSMA-TV, P =0.63; TL-PSMA, P =0.9). Findings on an entire tumor burden level were also corroborated in a hottest lesion analysis investigating the SUVmax of the most intense lesion per patient ( R 2 , 0.99; wCOV, 11.2%). Conclusion. In this prospective test-retest setting, SUV parameters demonstrated high repeatability, in particular in LNs, while volumetric parameters demonstrated low repeatability. Further, the large number of lesions and wide distribution of SUVs included in this analysis allowed for the demonstration of a dependence of repeatability on SUV, with higher SUVs having more robust repeatability.