Purpose To assess the diagnostic efficacy of 99mTc-sulfur colloid lymphoscintigraphy in chylothorax and chylous ascites, and the utility of single-photon emission computed tomography-computed tomography (SPECT/CT) in localizing the sites of leaks. Methods Data from patients who underwent lymphoscintigraphy for clinical suspicion of chylothorax or chylous ascites were retrospectively analyzed. Biochemical fluid analysis was taken as the reference standard. Pleural fluid triglyceride level > 110 mg/dL (with pleural fluid/serum ratio > 1) and a cholesterol level < 200 mg/dL (with pleural fluid/serum ratio < 1) were considered confirmatory for chylothorax. Ascitic fluid triglyceride level > 200 mg/dL with a low cholesterol level (ascites fluid/serum ratio < 1) was considered confirmatory for chylous ascites. Results 26 patients (15 males, 57.7%) aged 9 months to 68 years were enrolled in the study. Based on the reference standard, 17 had chylothorax or chylous ascites (9 with surgical history). Lymphoscintigraphy was positive in 16 (with 1 false positive) and negative in 10 (with 2 false negatives). The sensitivity, specificity, negative predictive value, positive predictive value, and accuracy of lymphoscintigraphy were 88.2% (63.6–98.5%), 88.9% (51.8–99.7%), 80.0% (51.6–93.8%), 93.8% (70.1–99.0%), and 88.5% (69.9–97.6%), respectively. SPECT/CT could localize sites of leaks in 61.5% (8/13) with a localization rate of 77.8% (7/9) and 25.0% (1/4) in patients with surgical and nonsurgical causes, respectively. Conclusion 99mTc-sulfur colloid lymphoscintigraphy is a highly efficacious noninvasive modality to diagnose chylothorax or chylous ascites with a high positive predictive value. SPECT/CT could localize the sites of leaks more frequently in patients with surgical causes.