Cancer is a genetic disease and thus is influenced by oncogenes and tumor suppressor genes. To determine whether the genetic analysis of pleural fluid can be used to diagnose malignant effusion, we investigated p53 and FHIT mutations and microsatellite alterations (MA) in the pleural fluid of 40 patients with pleural effusion associated with malignancy (ME) and in the pleural fluid of 17 patients with tuberculous pleurisy (TB) as a control group. p53 mutations were detected in five ME patients (13%) and in no TB patient, and FHIT mutations were detected in seven ME patients (18%) and two TB patients (12%). For four microsatellite markers, D3S1234, D3S1285, D9S171, and TP53, in ME patients, loss of heterozygosity (LOH) was seen in 10 (25%), 5 (13%), 10 (25%), and 6 patients (15%), respectively, and microsatellite instability (MI) in 6 (15%), 0 (0%), 1 (3%), and 3 patients (8%), respectively. Using the same markers, in TB patients, LOH was seen in three (18%), one (6%), three (18%), and one (6%), respectively, and MI in one (6%), zero (0%), zero (0%), and zero (0%), respectively. Twenty-five ME cases (63%) exhibited MA (LOH or MI) in at least one marker. Moreover, in four (80%) of five ME cases with negative cytology and no carcinoembryonic antigen increase in pleural fluid, MAs were identified. In ME, positive cytology was found in 42.5%, and positive MA, using four markers, in 63%. Although still limited in terms of sensitivity and specificity, this study shows that molecular diagnostic strategies could enhance the diagnostic yield in cases of malignant effusion.