Objectives The objective was to evaluate the diagnostic accuracy of five health literacy screening instruments in emergency department (ED) patients: the Rapid Evaluation of Adult Literacy in Medicine-Revised (REALM-R), the Newest Vital Sign (NVS), Single Item Literacy Screens (SILS), health numeracy, and physician gestalt. A secondary objective was to evaluate the feasibility of these instruments as measured by administration time, time on task, and interruptions during test administration. Methods This was a prospective observational cross-sectional study of a convenience sampling of adult patients presenting during March 2011 and February 2012 to one urban university- affiliated ED. Subjects were consenting non-critically ill, English-speaking patients over the age of 18 years without aphasia, dementia, mental retardation, or inability to communicate. The diagnostic test characteristics of the REALM-R, NVS, SILS, health numeracy, and physician gestalt were quantitatively assessed by using the short Test of Functional Health Literacy in Adults (S-TOFHLA). A score of 22 or less was the criterion standard for limited health literacy (LHL). Results A total of 435 participants were enrolled, with a mean (±SD) age of 45 (±15.7) years, and 18% had less than a high school education. As defined by an S-TOFHLA score of 22 or less, the prevalence of LHL was 23.9%. In contrast, the NVS, REALM-R, and physician gestalt identified 64.8, 48.5, and 35% of participants as LHL, respectively. A normal NVS screen was the most useful test to exclude LHL, with a negative likelihood ratio of 0.04 (95% confidence interval [CI] = 0.01 to 0.17). When abnormal, none of the screening instruments, including physician gestalt, significantly increased the posttest probability of LHL. The NVS and REALM-R require 3 and 5 minutes less time to administer than the S-TOFHLA. Administration of the REALM-R is associated with fewer test interruptions. Conclusions One-quarter of these ED patients had marginal or inadequate health literacy. Among the brief screening instruments evaluated, a normal NVS result accurately reduced the probability of LHL, although it will identify two-thirds of ED patients as high risk for LHL. None of the brief screening instruments significantly increases the probability of LHL when abnormal.