Background: Checklists have been shown to improve performance of complex, error-prone processes. To develop a checklist with potential to reduce the likelihood of diagnostic error for patients presenting to the Emergency Room (ER) with undiagnosed conditions. Methods: Participants included 15 staff ER physicians working in two large academic centers. A rapid cycle design and evaluation process was used to develop a general checklist for high-risk situations vulnerable to diagnostic error. Physicians used the general checklists and a set of symptom-specific checklists for a period of 2 months. We conducted a mixed methods evaluation that included interviews regarding user perceptions and quantitative assessment of resource utilization before and after checklist use. Results: A general checklist was developed iteratively by obtaining feedback from users and subject matter experts, and was trialed along with a set of specific checklists in the ER. Both the general and the symptom-specific checklists were judged to be helpful, with a slight preference for using symptom-specific lists. Checklist use commonly prompted consideration of additional diagnostic possibilities, changed the working diagnosis in approximately 10% of cases, and anecdotally was thought to be helpful in avoiding diagnostic errors. Checklist use was prompted by a variety of different factors, not just diagnostic uncertainty. None of the physicians used the checklists in collaboration with the patient, despite being encouraged to do so. Checklist use did not prompt large changes in test ordering or consultation. Conclusions: In the ER setting, checklists for diagnosis are helpful in considering additional diagnostic possibilities, thus having potential to prevent diagnostic errors. Inconsistent usage and using the checklists privately, instead of with the patient, are factors that may detract from obtaining maximum benefit. Further research is needed to optimize checklists for use in the ER, determine how to increase usage, to evaluate the impact of checklist utilization on error rates and patient outcomes, to determine how checklist usage affects test ordering and consultation, and to compare checklists generally with other approaches to reduce diagnostic error.