Morphological and genetic characters of male and female adults of Batocera lineolata Chevrolat were studied to determine intraspecific sex differences. Morphologically, the 9th and 10th segments of the antennae of male adults have odontoid processes that the female lacks. The longitudinal stripes of each abdominal segment of female adults appear to be connected between each adjacent segment, but those of the male do not appear continuous. Female adults also have a narrow V-shaped longitudinal groove in the 5th abdominal segment, whereas males do not. Amplification of the mitochondrial cytochrome c oxidase subunit I gene, cytochrome c oxidase subunit II gene, cytochrome b gene, and ribosomal 16S rRNA gene of four mitochondrial DNA showed sequences that differed between male and female adults. The similarity of the four genes between male and female adults is 98.7%, 99.1%, 98.4%, and 98.8%, respectively. The A+T contents of the four genes in female adults were all higher than in male adults. The difference in content of A+T versus C+G base pairs in female adults was higher than in males. This method of combined morphological and genetic analysis appears to be an accurate and straightforward tool for distinguishing male and female adults of B. lineolata.