There is accumulating evidence that soluble amyloid‐β (Aβ) oligomers, rather than amyloid fibrils, are the principal pathogenic species in Alzheimer disease (AD). Here, we have developed a novel enzyme‐linked immunosorbent assay (ELISA) specific for high‐molecular‐weight (HMW) Aβ oligomers. Analysis of Aβ oligomers derived from synthetic Aβ 1‐42, by size‐exclusion chromatography (SEC), revealed that our ELISA specifically detected HMW Aβ oligomers of40–200 kDa. Using this ELISA, we detected significantly higher (P<0.0001) signals in cerebrospinal fluid (CSF) samples from 25 patients with AD or mild cognitive impairment (MCI), compared to 25 age‐matched controls. As a test for discriminating between the AD/MCI and control groups, the area under the curve in receiver operating characteristic analysis for the CSF HMW Aβ oligomers was greater than that for CSF Aβx‐42. Furthermore, the CSF levels of HMW Aβ oligomers showed a negative correlation with Mini‐Mental State Examination scores in the AD/MCI group. We conclude that the CSF HMW Aβ oligomers detected by our ELISAcould be useful as a diagnostic marker for AD, and also as a potential surrogate marker for disease severity. Our results support the idea that soluble HMW Aβ oligomers play a critical role in the pathogenesis and progression of AD.—Fukumoto, H., Tokuda, T., Kasai, T., Ishigami, N., Hidaka, H., Kondo, M., Allsop, D., Nakagawa, M. High‐molecular weight β‐amyloid oligomers are elevated in cerebrospinal fluid of Alzheimer patients. FASEB J. 24, 2716–2726 (2010). www.fasebj.org