<p>Understanding how micro-geographic isolation and multi-layered drivers shape intraspecific genetic structure is critical for conserving narrow-range cliff endemics. <i>Oreocharis guileana</i>, an endangered cliff-restricted herb endemic to Hong Kong–Shenzhen coastal mountains, helps clarify combined influences of postglacial climate change and urban fragmentation on population genetics. Using chloroplast haplotypes and genome-wide SNPs from 106 individuals across six populations (subpopulations) at four distinct geographic sites, we detected extremely low nucleotide diversity: Hong Kong subpopulations (π ≈ 1.5 × 10⁻⁴) outperformed Shenzhen populations (π ≈ 1.2 × 10⁻⁴), and positive Tajima’s D across all samples indicated historical bottlenecks. Strong genetic differentiation existed between Hong Kong and Shenzhen groups (<i>F</i>_<i>ST</i> = 0.219–0.243), with moderate divergence among three Shenzhen populations (<i>F</i>_<i>ST</i> = 0.072–0.103), and no obvious isolation-by-distance pattern. Ancestral split of the two regional lineages happened ~ 28,000 years ago owing to postglacial cliff habitat shrinkage, whereas population differentiation within Shenzhen arose the past two millennia alongside intensive human landscape alteration. Limited pollen and seed dispersal intrinsically restricts gene flow, and modern urbanization further strengthens isolation. We propose a hierarchical conservation framework: Hong Kong populations serve as core genetic reservoirs, the haplotype-unique SZ2 receives key protection, and improving connectivity among Shenzhen populations is superior to risky cross-border assisted gene flow. This study exposes urbanization-related genetic erosion risks for cliff-endemic plants and provides targeted conservation guidance for <i>O. guileana</i>.</p>