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The corolla tube length of the bumblebee-pollinated plant Isodon umbrosus shows conspicuous geographical variation, corresponding with the proboscis length of its bumblebee pollinators across its distributional range. We hypothesized that altitudinal variation in the pollinator assemblage is a principal factor mediating morphological and genetic differentiation among I. umbrosus populations. We examined determinants of the morphological and genetic differentiation of Isodon umbrosus by analyzing floral morphology and allozyme variation across the distributional range. A reanalysis of previous data confirmed that altitude was a good indicator of pollinator assemblages. Corolla tube length was highly variable among the 15 study populations, and genetic differentiation among the populations (G(ST) = 0.360) was also highly significant. The differentiation in corolla tube length was explained by altitudinal difference, a proxy of the difference in pollinator assemblages. Genetic differentiation among populations also tended to be affected by the same factor, but statistical support was weak. To better understand the mechanisms responsible for morphological and genetic differentiation in I. umbrosus, we need to investigate altitudinally different populations over a narrower geographical scale.