Several studies have attempted to investigate how the brain codes emotional value when processing music of contrasting levels of dissonance; however, the lack of control over specific musical structural characteristics (i.e., dynamics, rhythm, melodic contour or instrumental timbre), which are known to affect perceived dissonance, rendered results difficult to interpret. To account for this, we used functional imaging with an optimized control of the musical structure to obtain a finer characterization of brain activity in response to tonal dissonance. Behavioral findings supported previous evidence for an association between increased dissonance and negative emotion. Results further demonstrated that the manipulation of tonal dissonance through systematically controlled changes in interval content elicited contrasting valence ratings but no significant effects on either arousal or potency. Neuroscientific findings showed an engagement of the left medial prefrontal cortex (mPFC) and the left rostral anterior cingulate cortex (ACC) while participants listened to dissonant compared to consonant music, converging with studies that have proposed a core role of these regions during conflict monitoring (detection and resolution), and in the appraisal of negative emotion and fear-related information. Both the left and right primary auditory cortices showed stronger functional connectivity with the ACC during the dissonant portion of the task, implying a demand for greater information integration when processing negatively valenced musical stimuli. This study demonstrated that the systematic control of musical dissonance could be applied to isolate valence from the arousal dimension, facilitating a novel access to the neural representation of negative emotion.