Church forests in Ethiopia are remnants of once continuous forests and provide a unique opportunity to investigate how human-driven isolation and small population sizes influence the genetic diversity and structure of tree species. This study investigated three indigenous tree species of high conservation priority in Ethiopia that constitute a considerable and important part of church forests: Afrocarpus gracilior, Juniperus procera, and Olea europaea subsp. cuspidata. A total of 237 A. gracilior, 466 J. procera, and 341 O. europaea trees were sampled across multiple church and natural/non-church reference forests to determine the genetic diversity and population structure using DNA markers. The level of diversity varied among species and between populations within species. Afrocarpus gracilior populations exhibited low genetic diversity, small effective population sizes, and high inbreeding, indicating vulnerability and limited suitability as standalone seed sources. Maintaining variability requires combining multiple populations. Juniperus procera and O. europaea populations showed high genetic diversity, but some showed moderate inbreeding and relatively low effective population sizes, highlighting the need for population-level prioritization in forest management and restoration. These findings emphasize that conservation and forest restoration strategies cannot be generalized across species. Species-specific investigations and tailored management approaches are essential for maintaining genetic diversity and supporting long-term forest resilience.