Background The diagnostic value of ascitic cholesterol in the differential diagnosis of ascites is controversial. Aim To investigate the diagnostic performance of ascitic cholesterol in the differential diagnosis of ascites. Methods Consecutive patients with new-onset ascites were enrolled prospectively. The pertinent data were collected from 629 patients with all forms of ascites. Results In the training cohort, determination of the ascitic cholesterol level was a highly effective method of distinguishing non-portal hypertension (NPH) from portal hypertension (PH). At the pre-determined cut-off value of 45 mg/dL, the sensitivity of ascitic cholesterol was superior to the serum-ascites albumin gradient (SAAG) in identifying NPH-related ascites; the area under the receiver operating characteristic curve was 0.945. In the patients misdiagnosed based on SAAG classification, the diagnostic accuracy of ascitic cholesterol was 69%. The ascitic cholesterol level showed excellent performance in identifying peritoneal lesions in patients with mixed ascites. Conclusion Ascitic cholesterol is an excellent measure for detecting NPH ascites and for identifying peritoneal lesions in mixed ascites. Thus, this simple and cost-effective measure should be determined in patients with new-onset ascites (; ChiCTR-DCD-15006907).