In ovo exposure to o,p′‐dichloro‐diphenyl‐trichloroethane (o,p′‐DDT) impairs reproduction by inducing malformation of the reproductive organs in birds, although the mechanism remains unclear. Here, we examined the effects of o,p′‐DDT on the development of the reproductive organs, the expression of genes controlling sexual differentiation, and the plasma concentrations of testosterone and estradiol in Japanese quail embryos. o,p′‐DDT‐containing sesame oil was injected into the yolk sac on Embryonic Day (E) 3 at a dose of 500, 2,000, or 8,000 μg per egg. On E15, the reproductive organs were observed; the gonads and Müllerian ducts (MDs) were sampled to measure the mRNA of steroidogenic enzymes, sex steroid receptors, anti‐Müllerian hormone (AMH), and AMH receptor 2 (AMHR2); blood samples were collected to assay plasma testosterone and estradiol levels; and the gonads were used for histological analysis. o,p′‐DDT dose‐dependently increased the prevalence of hypertrophic MDs in females and residual MDs in males. In female MDs, o,p′‐DDT dose‐dependently decreased estrogen receptor (ER) α, ERβ, and AMHR2 mRNA expression. o,p′‐DDT dose‐dependently induced left‐biased asymmetry of testis size, and ovary‐like tissue was found in the left testis after exposure to 8,000 μg per egg o,p′‐DDT, although asymmetric gene expression did not occur. o,p′‐DDT did not affect ovarian tissue but did decrease 17α‐hydroxylase/C17‐20 lyase mRNA expression and dose‐dependently increased ERβ mRNA expression. o,p′‐DDT decreased plasma testosterone concentrations in females. These findings suggest that o,p′‐DDT induces hypertrophy of the MDs and ovarian tissue formation in the left testis. Abnormal MD development may be linked to altered gene expression for sensing estrogens and AMH signals.