To determine the usefulness of parathyroid scintigraphy in histological estimation for secondary hyperparathyroidism (2HPT) using Tc-99m sestamibi or Tc-99m tetrofosmin. Tc-99m sestamibi (MIBI) and Tc-99m tetrofosmin (Tetro) parathyroid imaging following double-phase study, magnetic resonance imaging (MRI), and ultrasound were performed on 14 patients with 2HPT. All patients underwent parathyroidectomy. The uptake of two tracers in parathyroid areas was compared with the histopathologic findings. Forty-nine parathyroid glands were surgically explored and histologically proven to be hyperplastic. Of these, 42 were diagnosed with nodular type (N-type) hyperplasia, and 7 with diffuse type (D-type) hyperplasia. MIBI and Tetro parathyroid imagings detected 34 and 35 parathyroid glands, respectively. The sensitivity of MIBI was determined to be 76.2% (32/42) for N-type, and 28.6% (2/7) for D-type. The sensitivity of Tetro was determined to be 78.6% (33/42) for N-type and 28.6% (2/7) for D-type. The sensitivity of both MIBI and Tetro was significantly higher for N-type than for D-type, 76.2% (32/42) vs. 28.6% (2/7) in MIBI, P = 0.022; 78.6% (33/42) vs. 28.6% (2/7) in Tetro, P = 0.015. The sensitivity of MRI was determined to be 76.2% (32/42) for N-type and 42.9% (3/7) for D-type, and the sensitivity of ultrasound was 71.4% (30/42) for N-type and 71.4% (5/7) for D-type. There was no significant difference in the sensitivity of MRI or ultrasound between N-type and D-type. The uptake ratios of MIBI and Tetro were also greater for N-type than for D-type. The detectability of both MIBI and Tetro was greater for N-type than for D-type. Tc-99m MIBI or Tc-99m Tetro parathyroid scintigraphy therefore may be used clinically to distinguish N-type from D-type parathyroid gland hyperplasia.