The Postpartum Bonding Questionnaire is a self-report questionnaire designed to screen disorders of the mother-infant relationship, which has been adapted to several countries. The aim of this study was to investigate validity evidence of the Brazilian version of the Postpartum Bonding Questionnaire (PBQ-Br) based on its internal structure (dimensionality, reliability, and measurement invariance between mothers with and without depressive symptoms) and on relations to other variables (depression). The present study is part of the Ribeirão Preto and São Luís Brazilian Cohort Studies, encompassing data collected from February 2011 to September 2013. Data were available for 2207 mothers from the general population of Ribeirão Preto and São Luís cities, who were assessed using the PBQ and the Edinburgh Postnatal Depression Scale. Regarding internal structure, the results of confirmatory factor analyses did not support the original four-factor model, nor the alternative structures tested. Exploratory factor analysis suggested a one-factor model with eight items for the collected data, accounting for 38.7% of the total variance with good internal consistency (α = 0.83). Concerning validity evidence based on relations to other variables, the mean PBQ-Br score for mothers with depressive symptoms was significantly higher than for mothers with no depressive symptoms. In conclusion, we found good validity evidence and the PBQ-Br proved to be a useful tool for assessing the mother-infant relationship in the Brazilian general population rather than for its original purpose, i.e., assessing bonding disorders. The results also highlight the importance of developing cross-cultural studies in order to provide useful measures for specific populations.