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Background: Difficult temperament in infancy is a risk factor for forms of later internalizing and externalizing psychopathology, including depression and anxiety. A better understanding of the roots of difficult temperament requires assessment of its early development with a genetically informative design. The goal of this study was to estimate genetic and environmental contributions to individual differences in infant negative emotionality, their persistence over time and their influences on stability between 5 and 18 months of age. Method: Participants were 244 monozygotic and 394 dizygotic twin pairs (49.7% male) recruited from birth. Mothers rated their twins for negative emotionality at 5 and 18 months. Longitudinal analysis of stability and innovation between the two time points was performed in Mplus. Results: There were substantial and similar heritability (approximately 31%) and shared environmental (57.3%) contributions to negative emotionality at both 5 and 18 months. The trait’s interindividual stability across time was both genetically- and environmentally- mediated. Evidence of innovative effects (i.e., variance at 18 months independent from variance at 5 months) indicated that negative emotionality is developmentally dynamic and affected by persistent and new genetic and environmental factors at 18 months. Conclusions: In the first two years of life, ongoing genetic and environmental influences support temperamental negative emotionality but new genetic and environmental factors also indicate dynamic change of those factors across time. A better understanding of the source and timing of factors on temperament in early development, and role of sex, could improve efforts to prevent related psychopathology. [ABSTRACT FROM AUTHOR]