Spatially resolved omics technologies reveal context-dependent cellular regulatory networks in tissues of interest. Beyond transcriptome analysis, information on epigenetic traits and chromatin accessibility can provide further insights on gene regulation in health and disease. Nevertheless, compared to the enormous advancements in spatial transcriptomics technologies, the field of spatial epigenomics is much younger and still underexplored. In this study, we report laser capture microdissection coupled to ATAC-seq (LCM-ATAC-seq) applied to fresh frozen samples for the spatial characterization of chromatin accessibility. We first demonstrate the efficient use of LCM coupled to in situ tagmentation and evaluate its performance as a function of cell number, microdissected areas, and tissue type. Further, we demonstrate its use for the targeted chromatin accessibility analysis of discrete contiguous or scattered cell populations in tissues via single-nuclei capture based on immunostaining for specific cellular markers. Display omitted •Spatially resolved chromatin accessibility profiling with LCM-ATAC-seq•Laser capture microdissection and in situ tagmentation enables targeted tissue profiling•LCM-ATAC-seq can be combined with cellular staining to investigate target populations•Not only contiguous but also scattered cell populations can be analyzed by LCM-ATAC-seq Spatial epigenomics is still underexplored compared to the advanced evolution of spatial transcriptomics technologies. Different methods have been proposed recently, with distinct advantages and drawbacks concerning the achievable resolution, the possibility to overlay a morphological staining, and the overall applicability. In this study, we report the targeted use of LCM for the investigation of chromatin accessibility. This spatial methodology enables the analysis of scattered tissue cell populations at a mini-bulk resolution, with the possibility to integrate cellular or morphological stainings on the same processed section. Carraro et al. establish a complete workflow for the spatial characterization of chromatin accessibility in tissues of interest based on laser capture microdissection coupled to ATAC-seq. The technology can be easily combined with a morphological staining, allowing the targeting of both contiguous and scattered cell populations with a mini-bulk resolution.