This article examines the measurement equivalence of the Spanish Differentiation of Self Inventory for Adolescents (S-DSI-A) in adolescent populations across Spanish-speaking countries (N = 5,552), including Argentina, Chile, Ecuador, Mexico, Peru, and Spain. Within the Bowen family system theory, Differentiation of self is one of the most recognized constructs to systemic researchers and couple and family therapy practitioners, with implications in relationship adjustment or mental and physical health. The study uses multigroup confirmatory factor analysis (MGCFA) to test for configural, metric, and scalar invariance of the S-DSI-A across the six samples. The results indicate that the S-DSI-A demonstrates configural and metric invariance, but scalar invariance is not achieved. These findings have implications for cross-cultural research on differentiation of self and highlight the importance of considering measurement equivalence when comparing results across different populations. Specifically, our results suggest that the S-DSI-A reliably assesses the latent variables of Emotional Reactivity (ER) and Emotional Cutoff (EC) uniformly across Spanish-speaking nations, but the mean scores for the ER and EC lack cross-sample comparability.