BACKGROUND Primary hyperparathyroidism (pHPT) is caused by single- or multiglandular disease (MGD). Patients with MGD have an increased risk of complications at surgery and for persistence and recurrence after surgery. The study evaluated whether preoperative clinical and biochemical characteristics could predict MGD in patients with pHPT. METHODS We retrospectively evaluated patients operated 1989-2013 for first-time, non-hereditary pHPT. MGD was defined in patients with more than one pathological gland excised at surgery or with persistent hypercalcemia after the excision of a single pathological parathyroid gland, confirmed by histopathology. Clinical and biochemical variables were compared in patients with single- and multiglandular disease. Logistic regression was used to identify variables predicting MGD, yielding odds ratios (OR) with 95% confidence intervals (CI). RESULTS There were 707 patients, of which 79 (11%) had MGD. Patients with MGD were more likely to have negative sestamibi scintigraphy than patients with single-gland disease, 15 of 49 (31%) vs. 70 of 402 (17%; p = 0.03), to suffer from diabetes (12 of 74, 16%) vs. 45 out of 626 patients (7.2%; p < 0.01) and had lower preoperative levels of urinary calcium (3.80 vs. 4.44 mmol/L; p = 0.04). Multivariable analysis identified negative scintigraphy (OR 2.42; 95% CI 1.18 to 4.79), diabetes (OR 2.75; 95% CI 1.31 to 4.97) and elevated levels of osteocalcin (OR 3.79, 95% CI: 1.75 to 8.21) as predictors of MGD. CONCLUSIONS Negative sestamibi scintigraphy, diabetes and elevated osteocalcin levels were predictors of MGD.