BACKGROUND AND PURPOSE: Imaging biomarkers are increasingly used to provide a better understanding of the pathophysiology of acute ischemic stroke. However, this approach of routinely using imaging biomarkers to inform treatment decisions has yet to be translated into successful randomized trials. The aim of this study was to systematically review the use of imaging biomarkers in randomized controlled trials in patients with acute ischemic stroke, exploring the purposes for which the imaging biomarkers were used. MATERIALS AND METHODS: We performed a systematic review of imaging biomarkers used in randomized controlled trials of acute ischemic stroke, in which a therapeutic intervention was trialed within 48 hours of symptom onset. Data bases searched included MEDLINE, EMBASE, strokecenter.org, and the Virtual International Stroke Trials Archive (1995-2014). RESULTS: Eighty-four studies met the criteria, of which 49 used imaging to select patients; 31, for subgroup analysis; and 49, as an outcome measure. Imaging biomarkers were broadly used for 8 purposes. There was marked heterogeneity in the definitions and uses of imaging biomarkers and significant publication bias among post hoc analyses. CONCLUSIONS: Imaging biomarkers offer the opportunity to refine the trial cohort by minimizing participant variation, to decrease sample size, and to personalize treatment approaches for those who stand to benefit most. However, within imaging modalities, there has been little consistency between stroke trials. Greater effort to prospectively use consistent imaging biomarkers should help improve the development of novel treatment strategies in acute stroke and improve comparison between studies.