Background <p>Renal fibrosis is strongly correlated with renal functional outcomes. Therefore, this is a significant finding in determining renal prognosis. There are various reports on the imaging evaluation of renal fibrosis, but these are not well established. Scanning acoustic microscopy (SAM) uses ultra-high-frequency ultrasound to visualize tissues in just over a minute. SAM can simultaneously measure acoustic data such as speed of sound (SOS). SOS indicates the elasticity (stiffness) of a material. In this study, we aimed to compare and evaluate SAM acoustic intensity images and SOS data with light microscopy images of renal lesions, especially renal fibrosis.</p> Methods <p>Renal specimens containing fibrosis were selected. The acoustic intensity images were compared to PAS-stained images. SOS data of the tubulointerstitium were compared with Masson’s trichrome (MT)-stained images. The blue intensity of MT staining, which indicates fibrosis, was numerically valued using image-processing software. Furthermore, the correlations between it and the SOS values were evaluated.</p> Results <p>The acoustic intensity images suggested tubular atrophy and interstitial expansion in the same areas as in the PAS staining. SOS values of interstitial expansion with fibrosis were higher than normal area, interstitial expansion without fibrosis. A weak positive correlation was observed between the SOS values and the blue intensity of MT staining.</p> Conclusions <p>SOS data can be used to evaluate renal fibrosis. The combination of SOS data and MT-stained images enables a more detailed evaluation of renal fibrosis. This study can contribute to the evaluation of renal fibrosis and has potential clinical applications in the future.</p>