HiPET is a recently developed prototype preclinical PET scanner dedicated to high sensitivity and high resolution molecular imaging. The HiPET system employs a phoswich depth of interaction (DOI) detector design, which also allows identification of the large majority of the cross layer crystal scatter (CLCS) events. This work evaluates its performance characteristics following the National Electrical Manufacturers Association (NEMA) NU4-2008 protocol.The HiPET consists of twenty flat panel type detectors arranged in two rings. The inner diameter is 160 mm and the axial field of view (FOV) is 104 mm. Each detector is comprised of two layers of phoswich scintillator crystal arrays, a tapered, pixelated glass lightguide and a multi anode photomultiplier tube (MAPMT). The front (gamma ray entrance) layer is a 48 × 48 pixelated cerium doped lutetium yttrium orthosilicate (LYSO) scintillator array with individual crystals measuring 1.01 × 1.01 × 6.1 mm. The back (towards the PMT) layer is a 32 × 32 pixelated bismuth germanate (BGO) scintillator array with individual crystals measuring 1.55 × 1.55 × 8.9 mm.For energy windows of 250–650 keV and 350–650 keV, the peak absolute sensitivity at the center of the FOV was 13.5% and 10.4% including CLCS events, and 11.8% and 8.9% excluding CLCS events, respectively. The average detector energy resolution derived by averaging the individual crystal spectra was 11.7% ± 1.4% for LYSO and 17.0% ± 1.4% for BGO. The 3D ordered-subsets expectation maximization (OSEM) reconstructed image of a point source in air, ranged from 0.73 mm to 1.19 mm, with an average value of 0.93 ± 0.09 mm at all measured locations. The peak noise equivalent count rate (NECR) and scatter fraction were 179 kcps at 12.4 MBq and 6.9% for the mouse-sized phantom, and 63 kcps at 11.3 MBq and 18.3% for the rat-sized phantom. For the NEMA image quality phantom, the uniformity was 5.8%, and the spillover ratios measured in the water- and air-filled cold region chambers were 0.047 and 0.044, respectively. The recovery coefficients (RC) ranged from 0.31 to 0.92.These results and in vivo evaluation demonstrate that the HiPET can achieve high quality molecular imaging for biomedical applications.