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Objectives: Quantification of tau accumulation using positron emission tomography (PET) is critical for the diagnosis of Alzheimer's disease (AD). This study aimed to evaluate the feasibility of 18F-florzolotau quantification in patients with AD using a magnetic resonance imaging (MRI)–free tau PET template, since individual high-resolution MRI is costly and not always available in practice. Methods: 18F-florzolotau PET and MRI scans were obtained in a discovery cohort including (1) patients within the AD continuum (n = 87), (2) cognitively impaired patients with non-AD (n = 32), and (3) cognitively unimpaired subjects (n = 26). The validation cohort comprised 24 patients with AD. Following MRI-dependent spatial normalization (standard approach) in randomly selected subjects (n = 40) to cover the entire spectrum of cognitive function, selected PET images were averaged to create the 18F-florzolotau-specific template. Standardized uptake value ratios (SUVRs) were calculated in five predefined regions of interest (ROIs). MRI-free and MRI-dependent methods were compared in terms of continuous and dichotomous agreement, diagnostic performances, and associations with specific cognitive domains. Results: MRI-free SUVRs had a high continuous and dichotomous agreement with MRI-dependent measures for all ROIs (intraclass correlation coefficient ≥ 0.980; agreement ≥ 94.5%). Similar findings were observed for AD-related effect sizes, diagnostic performances with respect to categorization across the cognitive spectrum, and associations with cognitive domains. The robustness of the MRI-free approach was confirmed in the validation cohort. Conclusions: The use of an 18F-florzolotau-specific template is a valid alternative to MRI-dependent spatial normalization, improving the clinical generalizability of this second-generation tau tracer. Key Points: • Regional18F-florzolotau SUVRs reflecting tau accumulation in the living brains are reliable biomarkers for the diagnosis, differential diagnosis, and assessment of disease severity in patients with AD. • The18F-florzolotau-specific template is a valid alternative to MRI-dependent spatial normalization, improving the clinical generalizability of this second-generation tau tracer. [ABSTRACT FROM AUTHOR]