AIM: Characterizing tumor heterogeneity with textural indices extracted from 18F-fluorodeoxyglucose positron emission tomography (FDG PET/CT) is of growing interest in oncology. Several series showed promising results to predict survival in patients with head and neck squamous cell carcinoma (HNSCC), analyzing various tumor segmentation methods and textural indices. This preliminary study aimed at assessing the inter-observer and inter-segmentation method variability of textural indices in HNSCC pre-therapeutic FDG PET/CT. MATERIALS AND METHODS: Consecutive patients with HNSCC referred in our department for a pre-therapeutic FDG PET/CT from January to March 2016 were retrospectively included. Two nuclear medicine physicians separately segmented all tumors using 3 different segmentation methods: a relative standardized uptake value (SUV) threshold (40%SUVmax), a signal-to-noise adaptive SUV threshold (DAISNE) and an image gradient-based method (PET-EDGE). SUV and metabolic tumor volume were recorded. Thirty-one textural indices were calculated using LIFEx software (www.lifexsoft.org). After correlation analysis, selected indices' inter-segmentation method and inter-observer variability were calculated. RESULTS: Forty-three patients (mean age 63.8±9.3y) were analyzed. Due to a too small segmented tumor volume of interest, textural analysis could not be performed in 6, 11 and 15 cases with respectively DAISNE, 40%SUVmax and PET-EDGE segmentation methods. Five independent textural indices were selected (Homogeneity, Correlation, Entropy, Busyness and LZLGE). There was a high inter-contouring method variability for Homogeneity, Correlation, Entropy and LZLGE (p<0.0001 for each index). The inter-observer reproducibility analysis revealed an excellent agreement for 3 indices (Homogeneity, Correlation and Entropy) with an intraclass correlation coefficient higher than 0.90 for the 3 methods. CONCLUSIONS: This preliminary study showed a high variability of 4 out of 5 textural indices (Homogeneity, Correlation, Entropy and LZLGE) extracted from pre-therapeutic FDG PET/CT in HNSCC using 3 different contouring methods. However, for each method, there was an excellent agreement between observers for 3 of these textural indices (Homogeneity, Correlation and Entropy).