We examined the risk of atopic diseases in twins born after assisted reproduction. Data on atopic diseases and assisted reproduction in 9694 twin pairs, 3–20 years of age, from the Danish Twin Registry were collected via multidisciplinary questionnaires. The risk of atopic diseases in twins born after assisted reproduction was compared with the risk in twins born after spontaneous conception using logistic regression and variance components analysis. Children born after assisted reproduction did not have a different risk of atopic outcomes (adjusted odds ratios [95% confidence intervals] for asthma: 0.95 [0.85, 1.07], P = 0.403; hay fever: 1.01 [0.86, 1.18], P = 0.918; and atopic dermatitis: 1.02 [0.81, 1.11], P = 0.773 respectively) compared with children born after spontaneous conception. Assisted reproduction did not modify the heritability of atopic diseases. This study does not support an association between assisted reproduction and development of atopic diseases. This result must be confirmed in subsequent studies, preferably of singleton populations.