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Our aim was to adapt and validate a Brazilian Portuguese version of the Twin Relationship Questionnaire developed by Fortuna et al. (2010) and validated by H. Segal and Knafo-Noam (2019) in Israel. The respondents were 862 Brazilian mothers of twins ( N = 1724 twins) with mean age of 35 years (SD = 6.1). The majority of the sample lived in the Southeast (61.8%) or in the South (24.5%) of Brazil. We conducted a Multilevel Confirmatory Factor Analysis with the pair of twins as second level variable, and the five-factor structure (closeness, dependence, conflict, dominance, and rivalry) proposed by the original validation study of H. Segal and Knafo-Noam (2019) was confirmed. The final model retained 15 items out of 22 proposed in the original version of the questionnaire. Although the TRQ-BR has fewer items, the accuracy compared to the original questionnaire was maintained. Mixed Model Analysis (LMM) of TRQ scores were used to investigate twins’ relationships as a function of zygosity, age groups, and sex in order to provide evidence of convergent validity of the instrument. As expected, mothers perceived monozygotic twins (MZ) as more depedent than dizygotic twins (DZ). Furthermore, male twin pairs were considered more conflictive when compared to female twins. The present study showed that TRQ-BR is an adequate instrument for research purposes in the Brazilian population. It can also be useful for applied areas such as clinical and educational fields.