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Background and Purpose: Delivery of high precision radiotherapy lymph node boosts requires detailed information on the interfraction positional variation of individual lymph nodes. In this study we characterized interfraction positional shifts of suspected malignant lymph nodes for rectal cancer patients receiving long course radiotherapy. Furthermore, we investigated parameters which could affect the magnitude of the position variation., Materials and Methods: Fourteen patients from a prospective clinical imaging study with a total of 61 suspected malignant lymph nodes in the mesorectum, presacral, and lateral regions, were included. The primary gross tumor volume (GTV p ) and all suspected malignant lymph nodes were delineated on six magnetic resonance imaging scans per patient. Positional variation was calculated as systematic and random errors, based on shifts of center-of-mass, and estimated relative to either bony structures or the GTV p using a hierarchical linear mixed model., Results: Depending on location and direction, systematic and random variations (relative to bony structures) were within 0.6-2.8 mm and 0.6-2.9 mm, respectively. Systematic and random variations increased when evaluating position relative to GTV p (median increase of 0.6 mm and 0.5 mm, respectively). Correlations with scan time-point and relative bladder volume were found in some directions., Conclusions: Using linear mixed modeling, we estimated systematic and random positional variation for suspected malignant lymph nodes in rectal cancer patients treated with long course radiotherapy. Statistically significant correlations of the magnitude of the lymph node shifts were found related to scan time-point and relative bladder volume., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)