Elevation gradient is characterized by verticality, highly heterogeneous climatic conditions, which drive species performance and distribution. Although ultraviolet-B radiation (UV-B; 280–315 nm) is well known to have negative effects on plant growth, there has been no direct evidence that plants growing at higher elevations are more severely affected by UV-B, which is known to increase with elevation. Due to previous ecological studies limited to phenotype or physiological levels, we applied unique molecular and biochemical approaches to specifically identify UV-induced DNA damage, cyclobutane pyrimidine dimer level (CPD), which is the primary product of UV-B. The key findings demonstrate that (1) CPD level was higher and explained mainly by UV-B and not by other environmental factors. It suggests that plants growing at higher elevation naturally suffer more from UV-B. (2) UV-induced DNA damage significantly varied among a wide range of native species, and this variation was related to functional groups. DNA repair, rather than UV protection, dominated in UV-B tolerance in the field. Our findings also suggest that UV-B tolerance mechanisms varied among species under evolutionary trade-off and synergism. (3) Phenotypic plasticity to UV stress varied along elevational gradients: more constitutive in highland ecotypes, but more inducible in lowland ecotypes. These ecotypic differentiations suggest that local adaptation occurred in parallel. It also implies that constitutive defense to UV-B is necessary to inhabit higher elevations.