Purpose: Our goal was to evaluate a multi-pinhole (MPH) collimator which allows changing configurations for mouse imaging and rat imaging. Procedures: The collimator length can be adjusted from 5 cm for rat imaging to a maximum of 8 cm for mouse imaging. Projections of mouse- and rat-size phantoms were simulated with collimator length of 8 cm, and the rat-size phantom was additionally simulated with collimator length of 5 cm. Bias and noise were assessed in the reconstructed images. Three physical phantoms were used to evaluate the axial sampling and resolutions for one-, four-, and fivepinhole single photon emission computed tomography (SPECT). Images of three different-sized rodents were also acquired. Results: Simulations showed that for rat imaging, shorter collimator length provided an improved bias-noise trade-off compared to that of longer collimator length. Axial distortions were significantly reduced for MPH compared to single pinhole imaging. The smallest rods visible for mouse imaging and rat imaging were 1 and 1.6 mm, respectively, and their corresponding absolute sensitivities were 3.47% and 2.02% at the center field-of-view for 5-pinhole imaging. The count ratios were 1:3.78:4.42, respectively, for one-, four-, and five-pinhole for same acquisition time. Good image quality was observed in real animal studies. Conclusions: This collimator allows flexible single pinhole and MPH SPECT imaging for rodents, achieving high resolution and detection efficiency with minimal image artifacts. © Academy of Molecular Imaging and Society for Molecular Imaging, 2011.