The molecular subtypes of breast cancer correlate more strongly to prognosis, treatment response, and local recurrence than the traditional classifications based on histopathology. The ability to determine the subtype of breast tumors during surgery or biopsy in real time would provide physicians with new diagnostic capabilities to screen suspicious lesions, to perform high-precision surgery on malignant and premalignant lesions, and to personalize treatment for patients. This work studied the potential of using the molecular subtypes as natural biomarkers for characterizing breast cancer with high-frequency ultrasound (10–100 MHz). We hypothesized that high-frequency ultrasound would be able to detect variations in cell biomechanical properties due to mutations found in aggressive subtypes (e.g., basal-like and Her2 +). These mutations alter the expression levels of proteins that regulate the actin cytoskeleton, thereby modifying the biomechanical and thus ultrasonic properties of the cells. Pulse-echo measurements were acquired in vitro from breast cancer cell lines with different subtypes. The results showed that each cell line produced a unique ultrasonic spectral signature. One of the cell lines additionally exhibited changes over time, possibly due to dedifferentiation. Correlation of the results to other cell characterization methods will also be presented. [This work was supported by Utah Valley University.]