The increase in women’s labor market participation emphasizes the importance of understanding maternal separation anxiety, that is, the unpleasant maternal emotional state, due to the actual or anticipated short-term separation from the child. Drawing on the insights of the attachment and psychoanalytic perspectives, the Maternal Separation Anxiety Scale was developed to overcome existing measurement gaps. However, prior research did not replicate its original three-factor structure in the contemporary context and in other cultural settings, using large samples composed of mothers of (...) preschool children. This study aimed to examine the factorial structure of the MSAS in a sample of 597 Portuguese mothers of children aged 5–84 months who completed the questionnaire. The exploratory factor analysis conducted in subsample 1 revealed a four-factor structure: Maternal Negative Feelings, Beliefs about Exclusive Maternal Care, Need of Proximity, and Perceptions of Separation Benefits for Children. Confirmatory factor analyses conducted in subsample 2 revealed that the original three-factor structure revealed a poor fit, whereas the four-factor solution revealed an acceptable fit. As in previous studies, our findings report deviations from the original three-factor structure of the MSAS. Three of the newly identified factors seem to reflect specific sub-dimensions that originally guided item development in the MSAS, namely, maternal negative feelings, maternal attitudes about the value of exclusive maternal care, and the need of proximity with the child. The last factor appears to represent a refinement of original items pertaining to perceptions about separation effects for children. (shrink)