The principle of free flaps based on perforator vessels and the development of deep inferior epigastric perforator flap (DIEP) is currently applied in reconstructive microsurgery. However one of the main disadvantages is the operative complexity. Through anatomical study and clinical experience with DIEP flap in breast reconstruction, the intra-muscular path of the perforator vessels was comparatively studied, establishing the main anatomical parameters in order to favor procedure planning. Thirty DIEP flaps from fifteen fresh cadavers were used. The following parameters were measured: the number, location, and intramuscular course of the perforator vessels. In addition, an initial clinical study was performed in 31 patients using 35 DIEP flaps in breast reconstruction. The number, location and the intramuscular course of the perforators were measured. One hundred ninety one perforator vessels were detected (6.3 per flap). Thirty four percent were located in the lateral row and the rectilinear course was observed in 79.17 percent of these vessels. In the medial row, only 18.1% of the perforator vessels presented this configuration (p=0.001). Thirty-one patients underwent DIEP flap breast reconstruction with 26 immediate and 4 bilateral. In 22 flaps (62.8%), two perforators were used. In 25 flaps (71.4%) the lateral row perforators with a rectilinear course were observed. Mean operative time was seven hours and thirty-seven minutes. Two total flap lost and two partial flap were observed. The majority of the lateral row perforators presented a rectilinear intramuscular course, which is shorter in length compared to the medial row ones. This anatomical characteristic favors dissection with reduced operative time and vascular lesion morbidity, resulting in an important anatomical parameter for DIEP flap harvesting.