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The aim of this study was to investigate the prevalence of cross-gender behavior during childhood, to estimate the influence of genotype and environment on variation in cross-gender behavior, and to explore the association of cross-gender behavior with maternal ratings of behavior problems as indexed by the Internalizing and Externalizing scales of the Child Behavior Checklist (CBCL). Cross-gender behavior was assessed by two items from the CBCL: “behaves like opposite sex” and “wishes to be of opposite sex.” As part of an ongoing longitudinal study of the Netherlands Twin Registry, mothers were asked to complete the CBCL for their twins when they were 7 (n∼14,000 twins) and 10 years old (n∼8,500 twins). The prevalence of cross-gender behavior (as measured by maternal report of behaving like or wishing to be the opposite sex) was 3.2% and 5.2% for 7-year-old boys and girls, respectively, and decreased to 2.4% and 3.3% for 10-year-old boys and girls. Surprisingly, the prevalence rate of cross-gender behavior of girls with a male co-twin was lower than of girls with a female co-twin. At both ages, the similarity for cross-gender behavior was greater in monozygotic than in dizygotic twins pairs. Genetic structural equation modeling showed that 70% of the variance in the liability of cross-gender behavior could be explained by genetic factors, at both ages and for both sexes. Cross-gender behavior was associated with higher scores on Internalizing and Externalizing problems, both in boys and in girls.