Contrast-enhanced ultrasound (CEUS) imaging is a reliable noninvasive clinical method of detecting and characterizing hepatocellular carcinoma (HCC) lesions. In this study, high-frequency (40 MHz) CEUS imaging was applied to small animals. Specifically, high-frequency ultrasound liver imaging with albumin-shelled microbubbles was employed to characterize suspected HCC lesions that developed in five Hepatitis B virus X (HBx) transgenic mice at around 14 to 16 months of age. After injecting the contrast agent, the time-intensity curves (TICs) of suspected HCC lesions, vessels in suspected HCC lesions, and surrounding liver parenchyma tissues were measured for 30 minutes. In four of the five mice, the intensity of the TICs of the suspected HCC lesions was enhanced by a mean of 13.5 dB in the arterial phase (at around 10-30 seconds), with a rapid "washout" starting at the beginning of the portal phase. The peak intensity relative to the baseline also increased from 3.27 dB at 18 months to 23.74 dB at 22 months. On the other hand, the mean contrast between the suspected HCC lesion and the liver parenchyma increased by 6.6 dB, thus allowing clear detection of the HCC margin. Histopathology investigations confirmed the HCC development in these four mice. For the remaining mouse, the absence of the peak in the arterial phase was also consistent with the histopathology finding of dysplasia. These results demonstrated that high-frequency CEUS imaging is an effective preclinical tool for identifying HCC lesions in mice.