This report presents the anatomic studies, animal studies, and initial clinical experience that provide a basis for splitting pectoralis major muscle and myocutaneous flaps into independent segmental subunits. The anatomic study of 105 human cadaver pectoralis major muscle and myocutaneous units and their neurovascular supply indicated a consistent segmentation of muscle morphology, neurovascular supply, and myocutaneous territory. Three major independent segments (clavicular, sternocostal, and external) were identified. These findings provide the anatomic basis for segmentally splitting pectoralis major flaps, and the surgical technique that evolved is described. Segmentally split pectoralis flaps were experimentally tested in animals and subsequently used in 36 clinical reconstructions. The variety of forms of segmentally split pectoralis major flaps used in this series is described and analyzed. The technique of segmentally splitting flaps provides a method both for leaving innervated muscle segments in situ to preserve donor motor function and for deriving two independent flaps from one muscle.