Rab25, an epithelial-specific member of the Rab family of small GTPases, was previously shown to be overexpressed in ovarian/primary peritoneal serous carcinoma compared to malignant mesothelioma using gene expression arrays. The objective of this study was to validate this finding at the mRNA and protein level. Quantitative PCR analysis of 112 Müllerian serous carcinomas (84 effusions, 28 primary ovarian carcinomas) and 22 malignant mesotheliomas (19 effusions, 3 solid specimens) showed significantly higher RAB25 mRNA expression in the former tumor (p < 0.001). Immunohistochemical analysis of Rab25 protein expression in 245 effusions showed significantly higher expression of this protein in Müllerian serous carcinoma compared to malignant mesothelioma (189/209 vs. 12/36 positive tumors, respectively; p < 0.001). Immunostaining of 101 patient-matched solid Müllerian carcinoma specimens (34 primary carcinomas, 67 metastases) showed expression levels comparable to effusions (94/101 positive specimens; p > 0.05). Rab25 mRNA and protein expression levels in Müllerian carcinoma effusions did not correlate with overall or progression-free survival. Our data confirm that Rab25 effectively differentiates Müllerian carcinomas from malignant mesothelioma at the mRNA and protein level, suggesting a role in the diagnostic work-up of serosal cancers.