Understanding genetic diversity and population structure in wild Citrus relatives is crucial for conservation and crop improvement. Here, we examined genetic variation in Atalantia buxifolia from the island of Taiwan and Atalantia ceylanica from Sri Lanka using 21 transferable microsatellite (SSR) markers originally developed for Citrus. A total of 132 individuals from 13 populations were genotyped. Both species exhibited moderate levels of polymorphism, with A. buxifolia showing slightly higher allelic richness and heterozygosity than A. ceylanica. Analysis of molecular variance indicated that most genetic variation occurred within individuals (68% in A. buxifolia and 82% in A. ceylanica), while moderate population differentiation was detected (F_ST = 0.356 and 0.204, respectively). STRUCTURE, DAPC, PCoA, and F_ST analyses revealed distinct regional clustering in A. buxifolia, particularly in the Shoushan population, whereas populations of A. ceylanica were weakly structured. Monmonier’s analysis identified genetic barriers only in A. buxifolia, and BayesAss indicated high self-recruitment and localized gene flow in both species. Overall, these results suggest high within-population genetic diversity but limited connectivity among populations, shaped by geographic isolation and habitat fragmentation. Our findings provide a baseline for conservation planning in Atalantia populations and highlight the importance of maintaining habitat connectivity to preserve genetic resilience.