Background/Aim: T2 weighted magnetic resonance (MR) imaging is the gold standard for locally advanced rectal cancer (LARC) staging. The potential benefit of functional imaging, as diffusion-weighted MR (DWI) and positron emission tomography-computed tomography (PET-CT), could be considered for treatment intensification strategies. Dose intensification resulted in better pathological complete response (pCR) rates. This study evaluated the inter-observer agreement between two radiation oncologists, and the difference in gross tumor volume (GTV) delineation in simulation-CT, T2-MR, DWI-MR, and PET-CT in patients with LARC. Patients and Methods: Two radiation oncologists prospectively delineated GTVs of 24 patients on simul-CT (CT GTV ), T2-weighted MR (T2 GTV ), echo planar b1000 DWI (DWI GTV ) and PET-CT (PET GTV ). Observers' agreement was assessed using Dice index. Kruskal-Wallis test assessed differences between methods. Results: Mean CT GTV , T2 GTV , DWI GTV , and PET GTV were 41.3±26.9 cc, 25.9±15.2 cc, 21±14.8 cc, and 37.7±27.7 cc for the first observer, and 42.2±27.9 cc, 27.6±16.9 cc, 19.9±14.9cc, and 34.8±24.3 cc for the second observer, respectively. Mean Dice index was 0.85 for CT GTV , 0.84 for T2 GTV , 0.82 for DWI GTV , and 0.89 for PET GTV , representative of almost perfect agreement. Kruskal-Wallis test showed a statistically significant difference between methods (p=0.009). Dunn test showed there were differences between DWI GTV vs. PET GTV (p=0.040) and DWI GTV vs. CT GTV (p=0.008). Conclusion: DWI resulted in smaller volume delineation compared to CT, T2-MR, and PET-CT functional images. Almost perfect agreements were reported for each imaging modality between two observers. DWI-MR seems to remain the optimal strategy for boost volume delineation for dose escalation in patients with LARC.Standard treatment for locally advanced rectal cancer (LARC) is represented by neoadjuvant chemoradiotherapy (CRT) followed by organ preservation surgery. It is well known that clinical outcomes depend on the results of these treatments, in particular related to pathological complete response (pCR).Response to neoadjuvant CRT is dose-dependent with pCR rates reaching 20.4% using treatment intensification, when dose escalation above 60 Gy are delivered (1). Furthermore, both better dose distribution to the target as well as sparing the adjacent small bowel and other organs at risk (OARs) can be obtained with modern radiotherapy 424