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Introduction. Half a century since the creation of the International Association of Bryologists, we carried out a review to identify outstanding challenges and future perspectives in bryology. Specifically, we have identified 50 fundamental questions that are critical in advancing the discipline. Methods. We have adapted a deep-rooted methodology of horizon scanning to identify key research foci. An initial pool of 258 questions was prepared by a multidisciplinary and international working group of 32 bryologists. A series of online surveys completed by a broader community of researchers in bryology, followed by quality-control steps implemented by the working group, were used to create a list of top-priority questions. This final list was restricted to 50 questions with a broad conceptual scope and answerable through realistic research approaches. Key results. The top list of 50 fundamental questions was organised into four general topics: Bryophyte Biodiversity and Biogeography; Bryophyte Ecology, Physiology and Reproductive Biology; Bryophyte Conservation and Management; and Bryophyte Evolution and Systematics. These topics included 9, 19, 14 and 8 questions, respectively. Conclusions. Although many of the research challenges identified are not newly conceived, our horizon-scanning exercise has established a significant foundation for future bryological research. We suggest analytical and conceptual strategies and novel developments for potential use in advancing the research agenda for bryology.