Chloroplast DNA (cpDNA) is a valuable tool for studying plant population genetics and gene flow by pollen in conifers, particularly through the application of chloroplast DNA microsatellite markers (cpSSRs). This study focuses on Chilgoza pine (Pinus gerardiana L.), an economically and ecologically significant tree species in Afghanistan. Despite its importance, comprehensive genetic research on Chilgoza pine has been limited. To address this gap, we developed novel cpSSR markers based on the Chilgoza pine’s chloroplast genome to assess genetic diversity, population structure, and pollen dispersal in a population from Gardiz, Afghanistan. Needle samples from 199 trees across four subpopulations and two age cohorts (young and old) were collected and analyzed. Our findings revealed 27 chloroplast microsatellite markers, of which six exhibited polymorphism. Haplotype analysis identified 32 unique haplotypes, with one most prevalent haplotype. Genetic diversity analysis showed comparatively high levels of diversity, with no genetic differentiation between young and old tree cohorts. Fine-scale spatial genetic structure (FSGS) analysis revealed significant but weak family structure and relatedness in young cohorts, suggesting distance-dependent gene flow. Our study underscores the utility of cpSSRs in characterizing genetic diversity and structure, which is crucial for the conservation and sustainable management of Chilgoza pine forests. These findings provide insights for developing conservation strategies and highlight the importance of genetic marker studies to aid the preservation of biodiversity that supports local livelihoods.