Objectives:Quantify target volume delineation uncertainty for CT/MRI simulation and MRI-guided adaptive radiotherapy in rectal cancer. Define optimal imaging sequences for target delineation.Methods:Six experienced radiation oncologists delineated clinical target volumes (CTVs) on CT and 2D and 3D-MRI in three patients with rectal cancer, using consensus contouring guidelines. Tumour GTV (GTVp) was also contoured on MRI acquired week 0 and 3 of radiotherapy. A STAPLE contour was created and volume and interobserver variability metrics were analysed.Results:There were statistically significant differences in volume between observers for CT and 2D-MRI-defined CTVs (p < 0.05). There was no significant difference between observers on 3D-MRI. Significant differences in volume were seen between observers for both 2D and 3D-MRI-defined GTVp at weeks 0 and 3 (p < 0.05). Good interobserver agreement (IOA) was seen for CTVs delineated on all imaging modalities with best IOA on 3D-MRI; median Conformity index (CI) 0.74 for CT, 0.75 for 2D-MRI and 0.77 for 3D-MRI. IOA of MRI-defined GTVp week 0 was better compared to CT; CI 0.58 for CT, 0.62 for 2D-MRI and 0.7 for 3D-MRI. MRI-defined GTVp IOA week three was worse compared to week 0.Conclusion:Delineation on MRI results in smaller volumes and better IOA week 0 compared to CT. 3D-MRI provides the best IOA in CTV and GTVp. MRI-defined GTVp on images acquired week 3 showed worse IOA compared to week 0. This highlights the need for consensus guidelines in GTVp delineation on MRI during treatment course in the context of dose escalation MRI-guided rectal boost studies.Advances in knowledge:Optimal MRI sequences for CT/MRI simulation and MRI-guided adaptive radiotherapy in rectal cancer have been defined.