Contrast-enhanced ultrasound (CEUS) imaging has been a reliable clinical method of detecting three vascular contrast phases and characterizing focal liver lesions. Previous results were all from human (i.e., clinical studies). The main purpose of this study was to extend this to small animals and to investigate the feasibility of using CEUS in preclinical research. Specifically, high-frequency (40 MHz) ultrasound liver imaging with albumin-shelled microbubbles was employed to detect the three vascular contrast phases and characterize focal liver lesions that developed in thirteen Hepatitis B virus X (HBx) transgenic mice at around 14 to 16 months of age. Previous studies indicated that 90-100% incidence of hepatocellular carcinoma (HCC) was observed in HBx transgenic male mice. After injecting the contrast agent, the time-intensity curves (TICs) of focal liver lesions, vessels in focal liver lesions and surrounding liver parenchyma tissues were measured for 30 minutes. The peak of mean intensity relative to the baseline increased 7.36 dB (p<0.02). On the other hand, the mean contrast between the focal liver lesion and the liver parenchyma increased by 7.74 (p<0.05) dB, thus allowing clear detection of the lesion margin. Histopathology investigations confirmed the development of the lesion in these mice. In addition, guidelines of European Federation of Societies for Ultrasound in Medicine and Biology were followed as an attempt to characterize features of the TICs in mice. The arterial phase was defined as 2 to 60 seconds post contrast injection, and the parenchyma phase was defined as the time period from 10 to 30 minutes post contrast injection. Comparing the imaging with the pathology results, the sensitivity, specificity and accuracy of CEUS for the detection of malignant focal liver lesion in HBx transgenic mice were 91 %, 100% and 92%. These results demonstrated that high-frequency CEUS imaging potentially can be used for detecting the three vascular contrast phases of malignant focal liver lesions and characterizing malignant focal liver lesions in mice. Thus can be a valuable tool in preclinical research.