ObjectivesWorld Health Organization (WHO) guidelines for health programmes and healthcare delivery are the foundation of its technical leadership in public health and essential to decision-making globally. A key function of guideline development is to identify areas in which further evidence is needed because filling these gaps will lead to future improvements in population health. The objective of this study was to examine the knowledge gaps and research questions for addressing those gaps generated through the WHO guideline development process, with the goal of informing future strategies for improving and strengthening the guideline development process. Study designWe did a systematic, retrospective analysis of research questions identified in the published guidelines. MethodsWe analyzed guidelines published between January 1, 2008, and December 31, 2018, by the Communicable Diseases Cluster in five disease areas: tuberculosis (TB), HIV, malaria, TB-HIV, and neglected tropical diseases (NTDs). Research questions were extracted independently by two researchers. We analyzed the distribution of research questions by disease and by topic category and did a qualitative assessment of optimum practice for research question generation during the guideline development process. ResultsA total of 48 guidelines were included: 26 on HIV, 1 on malaria, 11 on TB, 5 on TB/HIV, and 5 on NTDs. Overall, 36 (75%) guidelines encompassed a total of 360 explicit research questions; the remainder did not contain specific research questions. The number of research questions that focused on TB was 49, TB/HIV was 38, HIV was 250, and NTDs was 23. The number of research questions that focused on diagnosis was 43 (11.9%) of 360, prevention was 62 (17.2%), treatment was 103 (28.6%), good practice was 12 (3.3%), service delivery was 86 (23.8%), and other areas was 54 (15%). Research questions were often not formulated in a specific or actionable way and were hard to identify in the guideline. Examples of good practice identified by the review team involved the generation of specific and narrowly defined research questions, with accompanying recommendations for appropriate study design. ConclusionsThe WHO must strengthen its approach to identifying and presenting research questions during the guideline development process. Ensuring access to research questions is a key next step in adding value to the guideline development process.