Two Atractylodes species, A. japonica Koidz. ex Kitam. (AJ) and A. macrocephala Koidz (AM) were used in this study. AJ population had higher amounts of Sesquiterpenoids and stronger tolerance to root rot but less vigor of root growth than AM population. Two populations (AJ and AM) were crossed to make interspecific hybrid population. A total of 98 lines propagated clonally were selected from a cross of AJ and AM, and evaluated for contents of sesquiterpenoids, atractylon (ATLN) and atractylenolide III (AT3) using high performance liquid chromatography (HPLC), and growth characters such as plant height, stem number and root weight. HPLC profiles of the hybrids were compared with those of parent plants, and it demonstrated the production of introgression hybrid by crossing between AJ and AM. Of 98 clonal lines,10 lines were selected by 10% level based on the growth vigor and tolerance to root rot, and AJM2102-51 line showed the heaviest root weight (117.1 g/plant) among them. A total of 98 hybrid lines contained on average 0.16 ± 0.10 mg/g of AT3, 2.00 ± 1.37 mg/g of ATLN, and 2.16 ± 1.40 mg/g of total sesquiterpenoids, showing high coefficients of variation (above 65%). Ten lines having high contents of sesquiterpenoids were selected, and AJM2101-15 had the highest amount (9.83 mg/g) of ATLN, and showed 40.8 g/plant of root weight similar to mean value (39.9 g/plant) of hybrid lines. The result showed that the introgression of both characters of vigorous growth from AM and high sesquiterpenoids content from AJ could be possible to make new hybrid lines by crossing between AJ and AM.