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Subterranean karst caves can contain unexpected biodiversity, but few studies related to spider population genetics have been conducted in the karst area of Southern China. In this study, we investigated the population genetic structure of <i>Trogloneta yunnanensis</i> (Song & Zhu, 1994) based on 73 spider samples from six underground populations in South China Karst. Population genetic structure analysis showed a clear divergence (<i>F</i>_ST > 0.9 and Nm < 0.05) among populations according to mitochondrial genes. The phylogenetic gene tree constructed by BI and ML methods recovered six geographic clades. Divergence time estimation indicated that the divergence of these six populations can be traced back to the late Pleistocene. We supposed that the geographic isolation led to the extreme population structure. According to this study and previous studies about troglobites living in this region, the subterranean habitats of the Yunnan-Guizhou Plateau may contain many organisms with similar genetic structures. The subterranean biodiversity in the karst area of Southern China needs to be re-evaluated and protected.