Introduction: Preoperative parathyroid adenoma localization allows for a minimally invasive surgical approach. Current methods used for adenoma localization include ultrasound, sestamibi scan, and occasionally magnetic resonance imaging (MRI). In cases in which an adenoma is not localized after a radiologic work-up, the surgeon must perform a four-gland exploration. Preoperative selective venous sampling (SVS) has been described as a safe and clinically effective method to localize a parathyroid adenoma after failed exploration. The purpose of this study was to evaluate the efficacy of SVS as a primary localization technique in cases where conventional localization techniques have failed. Materials and Methods: Fourteen patients with nonlocalizing parathyroid adenomas after ultrasonography, sestamibi scanning, and MRI were retrospectively reviewed. Eight patients underwent SVS prior to surgery and six patients underwent a four-gland neck exploration without preoperative SVS. The two groups were assessed for accuracy of the SVS in localizing the adenoma, operative time, length of hospitalization, and complications. Results: All of the patients in the study underwent successful uncomplicated surgical parathyroidectomy. The SVS proved accurate in preoperatively localizing the adenoma in 7 of 8 patients and allowed for a minimally invasive approach in 87% of patients. The mean operative time was 33 minutes for the SVS group and 67 minutes in the non-SVS group. None of the patients in either group experienced procedural complications. Conclusion: This study suggests that preoperative SVS may represent a safe and effective method of preoperatively localizing the nonlocalized parathyroid adenoma. In cases where conventional radiologic techniques fail to localize an adenoma, SVS may obviate the need for a four-gland exploration.