Purpose: Identification of suspicious PSMA-PET/CT-positive lymph node (LN) metastases (LNM) from prostate cancer (PCa) during lymphadenectomy (LA) is challenging. We evaluated an 111 In-labelled PSMA ligand (DKFZ-617, referred to as [ 111 In]PSMA-617) as a γ-emitting tracer for intraoperative γ-probe application for resected tissue samples in PCa patients. Forty-eight hours prior to LA, [ 111 In]PSMA-617 was administered intravenously in 23 patients with suspected LNM on PSMA-PET/CT (n = 21 with biochemical relapse, n = 2 at primary therapy). Resected tissue samples (LN, LNM and fibrofatty tissue) were measured ex situ by a γ-probe expressed as counts per second (CPS norm ). [ 111 In]PSMA-617 tissue sample uptake was measured by a germanium detector for verification and calculated as %IA lbm (percent injected activity per kilogram lean body mass at time of surgery). Based on a clinical requirement for a specificity > 95%, thresholds for both ex situ measurements were chosen accordingly. Correlation of the results from PET/CT, γ-probe and germanium detector with histopathology was done. Results: Eight hundred sixty-four LNs (197 LNM) were removed from 275 subregions in 23 patients, on average 8.6 ± 14.9 LNM per patient. One hundred four of 275 tissue samples showed cancer. Median γ-probe and germanium detector results were significantly different between tumour-affected (33.5 CPS norm , 0.71 %IA lbm ) and tumour-free subregions (3.0 CPS norm , 0.03 %IA lbm ) (each p value < 0.0001). For the chosen γ-probe cut-off (CPS norm > 23) and germanium detector cut-off (%IA lbm > 0.27), 64 and 74 true-positive and 158 true-negative samples for both measurements were identified. Thirty-nine and 30 false-negative and 6 and 5 false-positive tissue samples were identified by γ-probe and germanium detector measurements.Conclusion: [ 111 In]PSMA-617 application for LA is feasible in terms of an intraoperative real-time measurement with a γ-probe for detection of tumour-affected tissue samples. γ-probe results can be confirmed by precise germanium detector measurements and were significantly different between tumour-affected and tumour-free samples.