Can a longitudinally valid in vivo β-amyloid staging system be constructed for Alzheimer disease? In this multicenter longitudinal cohort study, a 4-level staging system using fluorine 18–labeled florbetapir positron emission tomography was defined using a combination of cerebrospinal fluid and positron emission tomography data. The β-amyloid stages had distinct associations with cerebrospinal fluid tau biomarkers, atrophy, and cognitive decline, had longitudinal validity in an analysis of transitions between stages, and were associated with distinct gene expression profiles; key results were validated in a replication cohort using fluorine 18-labeled flutemetamol positron emission tomography. Results of this study suggest that a novel β-amyloid staging system using positron emission tomography, in which stages are associated with different biological and clinically meaningful end points, can be used to track progression of Alzheimer disease longitudinally. This multicenter longitudinal cohort study of 741 participants uses in vivo β-amyloid cerebrospinal fluid and positron emission tomography findings to track progression of Alzheimer disease across 6 years of follow-up. Different brain regions appear to be involved during β-amyloid (Aβ) accumulation in Alzheimer disease (AD), but a longitudinally valid system to track Aβ stages in vivo using positron emission tomography (PET) is lacking. To construct a longitudinally valid in vivo staging system for AD using amyloid PET. Longitudinal multicenter cohort study using data accessed on August 20, 2018, from the Alzheimer’s Disease Neuroimaging Initiative database of scans performed from June 9, 2010, to July 12, 2018, from 741 persons: 304 without cognitive impairment, 384 with mild cognitive impairment, and 53 with AD dementia. Cerebrospinal fluid (CSF) Aβ42 and fluorine 18–labeled florbetapir ( 18 F-florbetapir) data were used to determine early, intermediate, and late regions of Aβ accumulation. β-Amyloid stages ranging from 0 to 3 were constructed using these composites. Each subsequent stage required involvement of more advanced regions. Patients were followed up at 2, 4, and 6 years. Replication and validation were conducted using an independent cohort (Swedish BioFINDER) and gene expression information from the Allen Human Brain Atlas database. Analyses were conducted August 21, 2018, to May 24, 2019. The main outcome was change in stage. Stages were compared for diagnosis, CSF biomarkers of tau, and longitudinal atrophy, cognitive measures, and regional gene expression. Transitions between stages were tested using longitudinal 18 F-florbetapir data. Among 641 participants with CSF Aβ42 data and at least two 18 F-florbetapir scans, 335 (52.3%) were male. The early region of Aβ accumulation included the precuneus, posterior cingulate, isthmus cingulate, insula, and medial and lateral orbitofrontal cortices. The late region included the lingual, pericalcarine, paracentral, precentral, and postcentral cortices. The intermediate region included remaining brain regions with increased accumulation rates. In 2072 PET scans from 741 participants, 2039 (98.4%) were unambiguously staged. At baseline, participants with stage 0 (n = 402) had a 14.7% (95% CI, 11.2%-18.1%) probability of progression to a higher stage; stage 1 (n = 21), 71.4% (95% CI, 50.0%-90.9%); and stage 2 (n = 79), 53.1% (95% CI, 42.2%-64.0%). Seven of the 741 participants (0.9%) reverted to a lower stage. Higher stages were associated with lower CSF Aβ42 concentrations (from stage 1 at baseline), greater CSF P-tau (from stage 1) and CSF T-tau (from stage 2), and accelerated cognitive decline (from stage 2) and atrophy (from stage 3), even when adjusting for clinical diagnosis. Key findings were replicated in the BioFINDER cohort (N = 474). The regions of different stages differed by gene expression profiles when using the transcriptome from the Allen Human Brain Atlas, especially involving genes associated with voltage-gated ion channel activity especially involving genes associated with voltage-gated ion channel activity, but also blood circulation, axon guidance, and lipid transportation. Results of this study suggest that this robust staging system of Aβ accumulation may be useful for monitoring patients throughout the course of AD. Progression through stages may depend on underlying selective vulnerability in different brain regions.