BACKGROUND: High quality health systems research (HSR) in fragile and conflict-affected states (FCAS) is essential to guiding the policies and programmes that will improve access to health services and, ultimately, health outcomes. Yet, conducting HSR in FCAS is challenging. An understanding of these challenges is essential to tackling them and to supporting research conducted in these complex environments. Led by the Thematic Working Group on Health Systems in FCAS, the primary aim of this study was to develop a research agenda on HSR in FCAS. The secondary aim was to identify the challenges associated with conducting HSR in these contexts. This paper presents these challenges. METHODS: Guided by a purposely-selected steering group, this qualitative study collected respondents' perspectives through an online survey (n = 61) and a group discussion at the Third Global Symposium on HSR in September 2014 (n = 11). Respondents with knowledge and/or experience of HSR in FCAS were intentionally recruited. RESULTS: Of those ever involved in HSR in FCAS (45/61, 75%), almost all (98%) experienced challenges in conducting their research. Challenges fall under three broad thematic areas: (1) lack of appropriate support; (2) complex local research environment, including access constraints, weak local research capacity, collaboration challenges and lack of trust in the research process; and (3) limited research application, including rapidly outdated findings and lack of engagement with the research process and results. CONCLUSIONS: This study shows that those familiar with HSR in FCAS face many challenges in gaining support for and in conducting and applying high-quality research. There is a need for more sustainable support, including commitment to and long-term funding of HSR in FCAS; investment in capacity building within FCAS to meet the challenges related to implementation of research in these complex environments; relationship and trust building among stakeholders involved in HSR, particularly between local and international researchers and between researchers and participants; and innovative and flexible approaches to research design and implementation in these insecure and rapidly changing contexts.