Training under high interference conditions through interleaved practice results in performance suppression during training but enhances long-term performance relative to repetitive practice involving low interference. Previous neuroimaging work addressing this contextual interference effect of motor learning has relied heavily on the blood-oxygen-level-dependent response using functional magnetic resonance imaging methodology resulting in mixed reports of prefrontal cortex recruitment under IP and RP conditions. We sought to clarify these equivocal findings by imaging bilateral PFC recruitment using functional near-infrared spectroscopy while discrete key (...) pressing sequences were trained under IP and RP schedules and subsequently tested following a 24-h delay. An advantage of fNIRS over the fMRI BOLD response is that the former measures oxygenated and deoxygenated hemoglobin changes independently allowing for assessment of cortical hemodynamics even when there is neurovascular decoupling. Despite slower sequence performance durations under IP, bilateral PFC oxygenated and deoxygenated hemoglobin values did not differ between practice conditions. During test, however, slower performance from those previously trained under RP coincided with hemispheric asymmetry in PFC recruitment. Specifically, following RP, test deoxygenated hemoglobin values were significantly lower in the right PFC. The present findings contrast with previous behavioral demonstrations of increased cognitive demand under IP to illustrate a more complex involvement of the PFC in the contextual interference effect. IP and RP incur similar levels of bilateral PFC recruitment, but the processes underlying the recruitment are dissimilar. PFC recruitment during IP supports action reconstruction and memory elaboration while RP relies on PFC recruitment to maintain task variation information in working memory from trial to trial. While PFC recruitment under RP serves to enhance immediate performance, it does not support long-term performance. (shrink)