High-frequency (HF) ultrasound imaging has been shown to be useful for non-invasively imaging anatomical structures of the eye and small animals in biological and pharmaceutical research, achieving superior spatial resolution. Cardiovascular research utilizing mice requires not only realtime B-scan imaging, but also ultrasound Doppler to evaluate both anatomy and blood flow of the mouse heart. This paper reports the development of an HF ultrasound duplex imaging system capable of both B-mode imaging and Doppler flow measurements, using a 64-element linear array. The system included an HF pulsed-wave Doppler module, a 32-channel HF B-mode imaging module, a PC with a 200 MS/s 14-bit A/D card, and real-time Lab View software. A 50 dB SNR and a depth of penetration of larger than 12 mm were achieved using a 35-MHz linear array with 50 μm pitch. The two-way beam widths were determined to be 165 to 260 μm and the clutter-energy-to-total-energy ratio (CTR) were 9.1 to 12 dB when the array was electronically focused at different focal points at depths from 4.8 to 9.6 mm. The system is capable of acquiring real-time B-mode images at a rate greater than 400 frames per second (fps) for a 4.8 × 13 mm field of view, using a 30 MHz 64-element linear array with 100 μm pitch. Sample in vivo cardiac high frame rate images and duplex images of mouse hearts are shown to assess its current imaging capability and performance for small animals.