Salivary glands give rise to approximately 30 histological distinct tumor types, which results in a diagnostic challenge for the pathologist. The present retrospective, immunohistochemical study aimed to evaluate the expression of Topoisomerase II-α, a nuclear enzyme, as a diagnostic and prognostic marker in benign and malignant salivary gland tumors, including leomorphic adenoma, mucoepidermoid carcinoma, acinic cell carcinoma and carcinoma ex-pleomorphic adenoma. A total of 59 cases of benign and malignant salivary gland tumors were included in the present study. Representative paraffin-embedded sections were immunostained for Topoisomerase II-α (Topo II-α). The expression level was semi-quantified for each case and then correlated with the histological diagnosis using hematoxylin and eosin-stained slides, grade of tumor and total survival. Significant differences were revealed between the expression level of Topo II-α in pleomorphic adenoma and mucoepidermoid carcinoma (P<0.001), carcinoma ex-pleomorphic adenoma (P<0.001), acinic cell carcinoma (P=0.005) and a group composed of all the malignant tumors (P<0.001). Cancer-specific survival rates were insignificantly increased in tumors expressing low levels of Topo II-α (P=0.464). Thus, the present study demonstrated different expression levels of Topo II-α in benign and malignant salivary gland tumors. These differing expression levels may act as valuable biomarkers for the correct histological diagnosis. Further studies conducted on a larger scale may lead to even more conclusive results.