Osteosarcoma is a common human malignancy with a high mortality rate worldwide. Recent studies have been focused on understanding the involvement of microRNA (miRNAs) in the pathogenesis of osteosarcoma. Therefore, the present study aimed to measure the expression levels of miR‐181a, cylindromatosis (CYLD), chromo box homolog 7 (CBX7), B‐cell lymphoma 2 (BCL2), and tumor protein p53 in tumor tissue and adjacent normal tissues in patients with osteosarcoma and its relationship with clinicopathological factors. The expression levels of miR‐181a, CYLD, CBX7, BCL2, and p53 were measured in 60 patients with osteosarcoma using quantitative real‐time polymerase chain reaction. Finally, we compared the relationship between these gene levels and clinicopathological factors in tumor and healthy tissues. Our results showed that the expression levels of miR‐181a, BCL2, and p53 were significantly higher in osteosarcoma tissue in comparison with normal tissues (p < .05). On the contrary, CYLD and CBX7 were downregulated in osteosarcoma tumor tissues compared to adjacent healthy tissues (p < .05). In addition, the expression levels of miR‐181a in tumor tissues were strongly correlated with patients' age, tumor size, clinical stage, cancer grade, and lymph node metastasis (p < .05). Our findings highlight new insights into understanding the role of miR‐181a in the pathogenesis of osteosarcoma. However, further studies are needed to elucidate miRNA as therapeutic targets for osteosarcoma.