Abstract Background Atractylodes lancea De Candolle is a medicinal plant distributed in East Asia. Its rhizome has been used as an important crude drug in traditional Chinese and Japanese medicines for the treatment of numerous diseases and disorders. In recent years, the demand for mass production of the crude drug with a stable quality has increased. Its major active compounds are sesquiterpenoids, such as β-eudesmol and hinesol that have closely related chemical structures with each other. As the criteria for evaluating the quality of A. lancea, the β-eudesmol/hinesol content ratio is considered important. In A. lancea, the ratio could be considered to be influenced by genetic factors, geographical environment factors and these interactions. Few studies of a detail genetic analyses for β-eudesmol/hinesol content ratio have been reported. Therefore, we evaluated the heritability and genotype–environment interaction on the β-eudesmol/hinesol content ratio in A. lancea using clonal lines propagated with division of rhizome. Results The heritability of the β-eudesmol/hinesol content ratio in A. lancea was evaluated through the cultivation of clonal lines of A. lancea in both different years (2016, 2017) and locations (Hokkaido, Ibaraki). Correlations between β-eudesmol and hinesol contents were identified in all clonal lines, with high correlation coefficients (r = 0.73–0.99). The broad-sense heritability of the β-eudesmol/hinesol content ratio was revealed to be high at 0.92. The effects of cultivation year were smaller than that of genotype, and few genotype–environment interactions were observed. In addition, the influence of cultivation location was also smaller than that of genotype, and the correlation between the two cultivation locations on the β-eudesmol/hinesol content ratio was high. The results suggested that the β-eudesmol/hinesol content ratio in A. lancea is highly dependent on genetic factors. Conclusion We demonstrate that the heritability of β-eudesmol/hinesol content ratio is high and that the effects of genetic factors were stronger than that of environmental factors such as cultivation location and year. Our findings suggested that selective breeding and clonal propagation are effective strategies for the production of A. lancea with stable qualities for use in the production of crude drugs.