AIM: To compare the results of 25 MHz and 50 MHz ultrasound biomicroscopy (UBM) regarding the image characteristics of the lens and its related diseases and to discuss the application value of 25 MHz UBM in ophthalmology. METHODS: A total of 302 patients (455 eyes) were included in this study from November 2014 to May 2015. Patient ages ranged from 5 to 89y (mean±SD: 61.0±17.7y). Different cross-sectional images of the lens were collected to compare and analyze the image characteristics and anterior segment parameters using 25 MHz and 50 MHz UBM in axial and longitudinal scanning modes, respectively. SPSS 19.0 for Windows, paired t-tests and B&A plot analysis were used for data analysis, and a value of P<0.05 was considered statistically significant. RESULTS: The 25 MHz UBM images displayed the lens shape more clearly than 50 MHz UBM images. Particularly for cataracts, the whole opacity of the lens was shown by 25 MHz UBM, but 50 MHz UBM only showed part of the lens. The means of the anterior segment parameters obtained using 25 MHz and 50 MHz UBM were as follows: central corneal thickness: 0.55±0.03 and 0.51±0.04 mm, respectively; central anterior chamber depth: 2.48±0.54 and 2.56±0.56 mm, respectively; and central lens thickness: 4.26±0.62 and 4.15±0.56 mm, respectively. A statistically significant difference was found between the results obtained with 25 MHz UBM and those obtained with 50 MHz UBM. The two devices had a good agreement in measuring the anterior segment parameters. CONCLUSION: The 25 MHz UBM had an obvious advantage in showing the lens shape. It can provide reliable imaging of the lens and its related diseases and has a high application value for ophthalmology.