OBJECTIVE: The aim of the present study was to evaluate the use of human epididymis protein 4 (HE4) and cancer antigen 125 (CA 125) biomarkers in differential diagnosis of malignant and benign endometrial tumours in a population of Czech women. DESIGN: Prospective study. SETTING: Department of Gynaecology and Obstetrics, Faculty of Medicine at Masaryk University and Faculty Hospital in Brno. METHODS: Our prospective study includes 115 patients with endometrioid adenocarcinoma and 106 patients with benign endometrial tumours in the control group. They were diagnosed with endometrial biopsy in the period from 7/2010 to 6/2013. The patients with cancer underwent definitive surgical treatment to determine the stage of disease. The median and ranges of serum levels were determined in relation to the histological result (benign vs malignant disease). Statistical analysis operates with logarithm values of markers because their distribution is not normal and uses logistic regression. RESULTS: While analysing two groups of patients with different histology, there was demonstrated a statistically significant difference (p < 0.05), only in HE4, by cut-off 48,5 pmol/l there was achieved sensitivity of 87.8%, specificity of 56.6% and negative predictive value of 81.1%. COCLUSION: Diagnostic benefit of HE4 can be considered especially in patients with increased risk of endometrial cancer and in patients with serious internal co-morbidities. HE4 could help in combination with clinical and ultrasound finding in the differentiation of prognostically various groups of patients and in decision-making in relation to the individualization of the treatment plan. However, the optimal cut-off for HE4 has not been solved yet, and to do so, it will require more research with larger studies and their comparative analysis..