Background: Relapse after initially successful treatment is a significant problem facing the treatment of opioid dependence. Evidence suggests craving elicited by re-exposure to drug cues may precipitate relapse. Attempts to identify neural biomarkers of cue-elicited craving have yielded inconsistent findings. We aimed to apply a novel continuous functional magnetic resonance imaging (fMRI) technique to follow the minute-to-minute evolution of brain responses which correlate with the waxing and waning of craving. Methods: Newly detoxified male opioid-dependent patients and healthy control participants attended two separate, counter-balanced, fMRI scanning sessions during which they viewed a 10-minute video (drug cue or neutral cue) followed by 5-minutes of fixation. Participants rated the intensity of their craving throughout each session. We hypothesised subcortical/ventral prefrontal cortex (PFC) regions and dorsal PFC regions would show different associations with craving reflecting their putative roles in appetitive processing versus cognitive control. Results: Compared with controls, drug cue (minus neutral cue) video recruited the left amygdala and was temporally correlated with craving. In contrast, dorsal anterior cingulate BOLD signal time-course was higher than controls only during a period after cue exposure when craving levels were declining. Against expectations neither the ventral striatum nor ventral PFC were significantly recruited by drug cue exposure. Conclusions: Findings suggest the amygdala has a central role in craving whereas the dorsal anterior cingulate may control craving in treatment-seeking patients. Time-course analysis yielded new insights into the neural substrates of craving that could objectively validate development of psychological and pharmacological approaches to sustained abstinence.