Species occupying heterogeneous ecological niches often exhibit pronounced phenotypic and genomic divergence, yet the mechanisms driving ecological differentiation remain unclear. Asian tree frogs (Rhacophorus sensu lato) span a vertical gradient from swamp, shrub, to arboreal habitats, providing an ideal system to investigate the evolutionary mechanisms underlying niche specialization. Here, we quantified interdigital webbing morphology in 630 individuals and generated genome-wide SNP data for 161 individuals representing 29 species. Our results show that webbing morphology is strongly correlated with vertical niche position. Phylogenomic analyses resolved ambiguous relationships and revealed a trend toward webbing reduction. Based on the clear phylogenetic relationships, introgression among distinct niches was inferred. Introgression was limited primarily between adjacent niche pairs (shrubby-swampy), while nearly complete absence of haplotype sharing across distinct niches indicates strong ecological barriers to gene flow. Selective sweep mapping identified 760 candidate genes exhibiting selection signals between arboreal and swampy groups, enriched in a wide range of pathways related to skeletal development (such as MAPK signaling) and cytoskeletal organization (such as Wnt signaling)—consistent with climbing and gliding adaptations. In contrast, long-term balancing selection preserved ancient polymorphisms to maintain functional allelic variation in genes associated with epithelial developmental plasticity (p63) and muscle architecture (obscn, ttn), with transposable elements potentially modulating regulatory variation. Our findings demonstrate that contrasting signatures of positive and balancing selection on functionally distinct genes drive the evolution of webbing morphology and locomotor traits, and limited gene flow across adjacent niches prevents homogenization, which together may contribute to adaptive diversification along vertical gradients.