Objectives: Post chemoradiotherapy (CRT) interval changes in apparent diffusion coefficient (ADC) have prognostic value in head and neck squamous cell cancer (HNSCC). The impact of using different region of interest (ROI) methods on interobserver agreement and their ability to reliably detect the changes in the ADC values was assessed. Methods: Following ethical approval, 25 patients (mean age 59.5 years, 21 male) with stage 3–4 HNSCC undergoing CRT were recruited for this prospective cohort study. Diffusion weighted MRI (DW-MRI) was performed pre-treatment and at 6 and 12 weeks following CRT. Two radiologists independently delineated ROIs using whole volume (ROI v), largest area (ROI a) or representative area (ROI r) methods at primary tumour (n = 22) and largest nodal (n = 24) locations and recorded the ADC mean. When no clear focus of increased DWI signal was evident at follow-up, a standardised ROI was placed (non-measurable or NM). Bland-Altman plots and interclass correlation coefficient (ICC) were assessed. Paired t-tests evaluated interval changes in pre- and post-treatment ADC mean at each location, which were compared to the smallest detectable difference (SDD). Results: Excellent agreement was obtained for all ROI methods at pre-treatment (ICC 0.94–0.98) and 6-week post-treatment (ICC 0.94–0.98). At 12-week post-treatment, agreement was excellent (ICC 0.91–0.94) apart from ROI r (ICC 0.86) and the NM nodal disease (ICC 0.87). There were significant interval increases in ADC mean between pre-treatment and post-treatment studies, which were greater than the SDD for all ROIs. Conclusions: ADC mean values can be reproducibly obtained in HNSCC using the different ROI techniques on pre- and post-CRT MRI, and this reliably detects the interval changes.