Longitudinal studies of mouse cancer models required large cohorts since autopsy was the only reliable method to evaluate treatment efficacy. This paper reports the use of high-resolution three-dimensional ultrasound micro-imaging to monitor prostate tumor development in genetically engineered mice. Twenty-nine genetically engineered prostate cancer mice, including castrated and uncastrated mice, were imaged by three-dimensional ultrasound. Qualitative comparisons of three-dimensional ultrasound images with histology sections of prostate tumors demonstrate the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. The correlation coefficient of tumor diameter measurements performed in vivo with three-dimensional ultrasound and at autopsy was 0.997. Prospective tumor detection sensitivity and specificity were 91.7% and 100%. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated diameter doubling times from 10 to 37 days for four prostate tumors during an initial period of rapid progression. Three-dimensional ultrasound will likely become the micro-imaging modality most readily adopted for mouse pre-clinical trial studies.