OBJECTIVE Molecular alterations in endometrial cancer have been shown to be prognostically significant but have not yet been implemented in the current clinical risk assessment. Few studies have investigated the reliability of molecular alterations in pre-operative specimens. Therefore, the objective was to determine whether molecular analysis of pre-operative endometrial cancer samples accurately reflects those alterations in the subsequent hysterectomy specimens. METHODS Paired pre-operative and hysterectomy specimens of 48 patients diagnosed with endometrial carcinoma, 42 endometrioid (EEC) and 6 non-endometrioid (NEEC) carcinomas, were analyzed for immunohistochemical expression of p53, PTEN and β-catenin. Tumor DNA was isolated and analyzed for microsatellite instability (MSI), TP53 mutations and somatic hot spot mutations in 13 genes. RESULTS In EEC patients, loss of PTEN, nuclear β-catenin and p53-mutant expression was found in 43%, 7% and 12%, respectively. No nuclear β-catenin was found in 5 of 6 NEEC patients, all serous cancers, whereas a p53-mutant expression was present in all serous cases. MSI was found in 19.5%, all EEC. Concordance for PTEN, β-catenin, p53 expression and MSI status was found in 79%, 92%, 79% and 93.5%, respectively. We detected 65 hot spot mutations in 39/48 (81%) tumors. Overall concordance of the GynCarta multigene analysis was 99.8%. CONCLUSIONS The results confirm the reliability of immunohistochemical and DNA-based techniques in the evaluation of molecular alterations in pre-operative endometrial specimens and high concordance rates with the definitive hysterectomy specimens. The resulting molecular signature provides initial pre-operative diagnostic information on the status of oncogenic pathways, which may contribute to individualized treatment strategies.