Researchers have long investigated emotion-related facial expressions, such as smiling and frowning, to further the field’s understanding of behavior, emotions, and psychopathology. Fewer studies have examined incongruent affect; facial expressions that do not match internal emotional experiences (e.g., smiling during frustration). Although not extensive, current accounts of incongruent affect in early childhood have assumed that these expressions indicate masking, an active regulatory process. Thus, many researchers contend that incongruent affect represents an adaptive emotion regulation strategy. However, little attention has been paid to incongruent affect, and its neurobiological correlates in early childhood. The present study examined complete versus partial incongruent smiling in preschool-aged children (3.5- to 5-years-old) who completed a frustration-eliciting task. We examined simultaneous neurobiological markers of incongruent smiles using functional near-infrared spectroscopy (fNIRS) and galvanic skin response (GSR) and tested links to parent-rated emotion regulation and psychopathology. Neutral and negative expressions were assessed as comparison expressions. Results revealed that complete incongruent smiling predicted poor effortful control, whereas partial incongruent smiling was not a significant predictor of regulation, neurobiological changes, or psychopathology. Neutral expressions predicted better effortful control skills and adaptive physiological response patterns. Results suggest that incongruent affect may signal poor regulation of positive affectivity in low-interpersonal contexts, while neutral expressions may act as masking expressions.