Effective exchange of individuals among populations (i.e. gene flow) is one crucial aspect to maintain genetic diversity and viability of populations, especially in the case of threatened species. Landscape composition and structure may facilitate or limit individual movement within and among populations. We used a landscape genetics (LG) approach to assess the connectivity patterns of the threatened Dupont’s lark (Chersophilus duponti duponti), considering their genetic patterns and the landscape features associated with its gene flow in Spain. We analysed genetic structure and relatedness based on 11 species-specific polymorphic microsatellites on 416 Dupont’s lark individuals sampled across peninsular Spain between 2017 and 2019, covering most of the European distribution of the species. To assess the relationship between the landscape configuration and the species gene flow, we estimated genetic distance at the individual level (Dps). Next, we built a set of environmental surfaces from two time points (1990 and 2018), based on factors (land use and topography) influencing individuals’ movement. We then obtained resistance surfaces from an optimization process on landscape variables, and LG analyses were done for single and composite surface models for each year separately. Our findings from both time points demonstrate, for the first time, that scatter or mosaic structured vegetation composed by low agricultural and tree cover and high presence of sclerophyllous shrubs favoured Dupont’s lark dispersal, while dense and continuous tree cover, as well as areas of intensive agriculture, were limiting factors. Our results suggest the importance of steppe habitat patches both for the species establishment and dispersal. Besides, our results provide key information to develop specific conservation measures, including focusing on conserving and restoring steppe habitats as scattered and/or mosaic structured vegetation that could warrant the connectivity and persistence of Dupont’s lark populations.