BackgroundTitle X-funded family planning clinics have been identified as optimal sites for delivery of pre-exposure prophylaxis (PrEP) for HIV prevention to U.S. women. However, PrEP has not been widely integrated into family planning services, especially in the Southern U.S., and data suggest there may be significant implementation challenges in this setting.MethodsTo understand contextual factors that are key to successful PrEP implementation in family planning clinics, we conducted in-depth qualitative interviews with key informants from 38 family planning clinics (11 clinics prescribed PrEP and 27 did not). Interviews were guided by constructs from the Consolidated Framework for Implementation Research (CFIR), and qualitative comparative analysis (QCA) was used to uncover the configurations of CFIR factors that led to PrEP implementation.ResultsWe identified 3 distinct construct configurations, or pathways, that led to successful PrEP implementation: (1) high “Leadership Engagement” AND high “Available Resources”; OR (2) high “Leadership Engagement” AND NOT located in the Southeast region; OR (3) high “Access to Knowledge and Information” AND NOT located in the Southeast region. Additionally, there were 2 solution paths that led to absence of PrEP implementation: (1) low “Access to Knowledge and Information” AND low “Leadership Engagement”; OR (2) low “Available Resources” AND high “External Partnerships”.DiscussionWe identified the most salient combinations of co-occurring organizational barriers or facilitators associated with PrEP implementation across Title X clinics in the Southern U.S. We discuss implementation strategies to promote pathways that led to implementation success, as well as strategies to overcome pathways to implementation failure. Notably, we identified regional differences in the pathways that led to PrEP implementation, with Southeastern clinics facing the most obstacles to implementation, specifically substantial resource constraints. Identifying implementation pathways is an important first step for packaging multiple implementation strategies that could be employed by state-level Title X grantees to help scale up PrEP.Supplementary InformationThe online version contains supplementary material available at 10.1186/s43058-023-00450-2.