Diet reconstruction using mesowear analysis has mainly been applied to extinct species and their paleoenvironments. Little is known regarding the effects of dietary differences on sympatric environments using this analysis and the limited existing knowledge from extant species may introduce errors when applied to fossil assemblages. I aimed to determine the sensitivity of mesowear analysis using extant ungulates with known diets. An interspecific comparison was conducted using wild populations of Japanese serow (Capricornis crispus, n = 37) and sika deer (Cervus nippon, n = 55) living in deciduous broad-leaved forest of the Nikko National Park, central Japan. One of the mesowear variables differed significantly between the two species (Fisher's exact test: p < 0.05). According to hierarchical cluster and principal component analyses, Japanese serow were classified as browsers, while sika deer as mixed feeders. Previous studies support these results; therefore, mesowear analysis can be used to detect dietary differences in sympatric species.