BackgroundKey populations (KPs) such as female sex workers (FSW), men who have sex with men (MSM), people who inject drugs (PWID), and their partners contribute more than a quarter (27.5%) of new HIV infection in Ghana. Oral pre-exposure prophylaxis (PrEP) can substantially reduce HIV acquisition among this group. While the available research indicates KPs willingness to take PrEP in Ghana, little is known about the position of policymakers and healthcare providers on the introduction of PrEP for KPs. MethodsQualitative and quantitative data were collected through a cross-sectional study from September to October 2017 in the Greater Accra (GA) and Brong-Ahafo (BA) regions of Ghana. In-depth interviews (IDIs) were conducted with 20 regional and national policymakers and 23 healthcare providers to explore their level of support for PrEP and their perspectives on challenges and issues to consider for oral PrEP implementation. The quantitative survey involved interviews with 409 healthcare providers to examine willingness to provide PrEP, challenges to PrEP roll-out and how it should be implemented. Thematic content analysis was used for the qualitative interviews descriptive analysis was conducted using frequencies of variables of interest for the quantitative analysis. ResultsPolicymakers and providers (95%) in both regions expressed strong support for introducing PrEP for KPs. Key concerns regarding oral PrEP introduction included potential for behavioral disinhibition, non-adherence and side effects of medication, cost and long-term financial implications, and stigma related to HIV and key populations. Participants stressed the need to integrate PrEP into existing services. While over three-fourths of providers indicated they would personally provide PrEP to HIV negative partners of HIV positive clients (87%), people who have multiple sex partners (83%), and FSWs (78%), a smaller percentage indicated they would provide PrEP to MSM (66%) and PWID (58%). ConclusionsPolicymakers and providers recognize the value of PrEP but have concerns about disinhibition, non-adherence, and cost. Therefore, a range of strategies are needed to address their concerns including: sensitization with providers to mitigate underlying stigma towards KPs, particularly MSM, integration of PrEP into existing services, and innovative strategies to improve continued use of PrEP.