Oribatid mites represent widespread soil microarthropods, yet their genetic diversity and evolutionary history remain largely unexplored. The present study aimed to assess the genetic variability of the family Damaeidae along a surface–cave ecological gradient. Sampling was conducted at 24 localities across eight geomorphological units in the Western Carpathians (Slovakia). At each locality, specimens were hand-collected in caves and at their entrances, while adjacent surface habitats were sampled using soil sieving. For molecular analyses, five surface- dwelling and three cave-associated species were selected. A total DNA was non-destructively extracted from the whole specimens and genetic differentiation was assessed using the mitochondrial cytochrome c oxidase subunit I (COI) gene. A major difference in genetic variation was observed between the two ecological groups. Phylogenetic analyses revealed clear species clustering, with cave-associated species effectively isolated within individual caves or populations, indicating restricted gene flow. The phylogenetic tree topology of populations of Belba clavigera Willmann, 1954 and Epidamaeus berlesei (Michael, 1898) from cave entrances reflected their geographic origin. A similar pattern was observed in Kunstidamaeus lengersdorfi (Willmann, 1932), a species occurring predominantly in the aphotic zones of caves, despite the absence of troglomorphic traits. In contrast, populations of surface-dwelling species – Damaeus auritus C.L. Koch, 1835, D. gracilipes (Kulczynski, 1902), D. riparius Nicolet, 1855, and K. tecticola (Michael, 1888) – from comparatively distant localities showed genetic intermixing, suggesting interpopulation connectivity in certain cases. Divergence time estimates indicate a Miocene origin of major Damaeidae lineages, followed by diversification during the Pliocene. Haplotype networks demonstrated localised haplotypes in cave populations, whereas surface populations exhibited higher haplotype diversity, likely reflecting environmental heterogeneity. In addition, potential cryptic lineages were identified in B. clavigera from Jasovská Cave and E. berlesei from the surroundings of Domica Cave, highlighting the evolutionary complexity of these mites in this region. Overall, our results provide new insights into the genetic structure and diversification of surface and subterranean lineages of damaeid mites in the context of the biogeographic history of the Western Carpathians, which are considered an important evolutionary center of obligate cave fauna in Europe.