The aim of the present study was to evaluate the efficacy of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in diagnosing mediastinal and intra-abdominal lymphadenopathies. A total of 154 patients with mediastinal and intra-abdominal lymphadenopathies were included in this retrospective study between February 2010 and March 2015. Malignancy was suspected in the patients as a result of imaging findings and EUS-FNAs were performed to confirm the diagnoses. EUS and EUS-FNA data, as well as hospital medical records, were reviewed. The accuracy of EUS-FNA was 90.8% for diagnosing malignancy and 85.6% for diagnosing benign lymphadenopathy. In combination with flow cytometry (FCM), the accuracy of EUS-FNA to determine lymphoma was 94.2%. Among the malignant lymphadenopathy cases, 80 were caused by metastasis, 19 by lymphoma and 1 by myeloid leukemia. In the 53 benign cases, EUS-FNA revealed a nonspecific inflammatory condition in 27 patients, tuberculosis in 21 patients and Castleman's disease in 5 patients. The factors revealed to be associated with malignant lymphadenopathy included the sex and age of patients, as well as the location and size of the enlarged lymph node. In particular, celiac axis lymphadenopathy was associated with malignancy (23.0% of cases of malignancy, vs. 3.8% of benign lymphadenopathy). EUS-FNA results additionally suggested that the malignant lymph nodes observed in celiac axis were more likely to result from lymphoma (42.1%; 8/19 cases) than metastasis (18.8%; 15/80 cases; P=0.039). By contrast, malignant lymph nodes observed in the mediastinum were more likely to be caused by metastasis (47.5%; 38/80 cases) than lymphoma (10.5%; 2/19 cases; P=0.004). The results of the present study suggested that EUS-FNA is accurate for differentiating between malignancy and benign lymphadenopathy. Therefore, EUS-FNA in combination with FCM analysis, as a minimally invasive and highly sensitive tool, should be routinely performed for the identification of lymphoma. Additionally, examining the enlarged celiac axis lymph nodes of elderly males, who exhibit an increased risk of malignancy, may be beneficial.