This study proposed to investigate the optimal selection of b-values in diffusion-weighted imaging for distinguishing malignant from benign mediastinal lymph nodes. Diffusion-weighted imaging with six b-values was performed on 35 patients at 1.5 T. Image quality score, signal-to-noise ratio, and relative contrast ratio of lymph node to chest muscle were compared between the diffusion-weighted images with a b-value up to 800 and 1000 s/mm2. Using a lower and an upper b-value in the range of 0–1000 s/mm2, eight apparent diffusion coefficient maps were obtained from a mono-exponential model. Receiver operating characteristic analysis was employed to evaluate the performance of the apparent diffusion coefficients for distinguishing malignant from benign mediastinal lymph nodes by using the area under the curve as a criterion.The mean image quality score and the relative contrast ratio showed no difference between b-values of 800 and 1000 s/mm2. In the receiver operating characteristic analysis, the areas under the curve of apparent diffusion coefficient with b-value pairs of (0, 800), (0, 1000), and (50, 800) s/mm2 were significantly higher than those from the other b-value pairs. No significant difference was observed among the three b-value pairs. Apparent diffusion coefficient obtained from b-value pairs of (0, 800), (0, 1000), and (50, 800) s/mm2 showed superior diagnostic performance compared to the other b-value combinations. Based on several practical considerations, the b-value pair of (50, 800) s/mm2 is recommended for differential diagnosis of mediastinal lymph nodes.