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The present study aimed to evaluate the influence of vertical location and spacing of perforators within flaps on the outcomes of deep inferior epigastric perforator (DIEP) flap breast reconstruction.Patients who underwent unilateral breast reconstruction with unipedicle DIEP flaps were identified. They were categorized into cohorts based on the entry of perforators in the middle third (cohort 1), upper third (cohort 2), and multiple third parts (cohort 3) of the flaps. Perfusion-related complications including fat necrosis diagnosed with ultrasound and donor-site morbidity were compared between the cohorts.A total of 287 patients were analyzed, including 51 in cohort 1, 75 in cohort 2, and 161 in cohort 3. The cohorts were well matched, except for a greater number of perforators and more frequent harvest of both medial and lateral row perforators in cohort 3. Rates of overall perfusion-related complications and fat necrosis differed significantly between cohorts, with the highest rates in cohort 2. Fat necrosis occurred predominantly in the caudal portion of the flap in cohort 2, whereas it was relatively evenly distributed in the cephalic and caudal portions in cohorts 1 and 3. Rates of donor-site complications were similar across the cohorts. Multivariate analyses demonstrated that vertical spacing of perforators had an independent influence on developing perfusion-related complications and fat necrosis, showing that cohort 2 had significantly higher odds compared with cohorts 1 and 3, respectively.Vertical spacing of perforators might affect the risk of perfusion-related complications in DIEP flap breast reconstruction.Therapeutic, III.