Population genetic diversity (GD) is essential for long-term adaptive potential of wild species and is shaped by life-history traits (LHTs) and microevolutionary processes. International conservation policy has endorsed the protection of GD with the adoption of a Headline Indicator in the 2022 Kunming-Montreal Global Biodiversity Framework: “the proportion of populations with an effective population size Ne above 500”. To shed light on the drivers of population Ne and improve GD indicator estimation from genetic data, we developed a categorization framework and estimated Ne for a selected set of 25 DNA datasets with rich metadata. The species represented with these datasets harbour contrasted LHTs strategies that relate to lifespan (axis 1), the spread of reproductive stages throughout lifespan (axis 2), and, additionally, to spatial genetic structure, dispersal capacity or population history (axis 3). For each species, we compared Ne estimates obtained from two methods, based on the linkage disequilibrium in populations, or based on a Bayesian method including various genetic summary statistics as prior information. We also considered technical data features such as the type of molecular marker, and the number and quality of SNPs. Our results show a wide range of Ne estimates within some species (including populations with Ne below the 500 threshold and others above it), and also among species within the same categories of LHTs. We discuss challenges for obtaining reliable DNA-based Ne estimates for GD indicators in species with contrasted life-history trait variation, life cycle and evolutionary scenarios. By exploring these challenges, we aim to contribute to developing best practices for GD indicator estimation for nature managers.Read less <