In vivo pre-clinical single-photon emission computed tomography (SPECT) is a valuable tool for functional small animal imaging, but several physical factors, such as scatter radiation, limit the quantitative accuracy of conventional scintillation crystal-based SPECT. Semiconductor detectors such as CZT overcome these deficiencies through superior energy resolution. To our knowledge, little scientific information exists regarding the accuracy of quantitative analysis in CZT-based pre-clinical SPECT systems for different isotopes. The aim of this study was to assess the quantitative accuracy of CZT-based pre-clinical SPECT for four isotopes: 201Tl, 99mTc, 123I, and 111In. The quantitative accuracy of the CZT-based Triumph X-SPECT (Gamma-Medica Ideas, Northridge, CA, U.S.A.) was compared with that of a conventional SPECT using GATE simulation. Quantitative errors due to the attenuation and scatter effects were evaluated for all four isotopes with energy windows of 5%, 10%, and 20%. A spherical source containing the isotope was placed at the center of the air-or-water-filled mouse-sized cylinder phantom. The CZT-based pre-clinical SPECT was more accurate than the conventional SPECT. For example, in the conventional SPECT with an energy window of 10%, scatter effects degraded quantitative accuracy by up to 11.52%, 5.10%, 2.88%, and 1.84% for 201Tl, 99mTc, 123I, and 111In, respectively. However, with the CZT-based pre-clinical SPECT, the degradations were only 9.67%, 5.45%, 2.36%, and 1.24% for 201Tl, 99mTc, 123I, and 111In, respectively. As the energy window was increased, the quantitative errors increased in both SPECT systems. Additionally, the isotopes with lower energy of photon emissions had greater quantitative error. Our results demonstrated that the CZT-based pre-clinical SPECT had lower overall quantitative errors due to reduced scatter and high detection efficiency. Furthermore, the results of this systematic assessment quantifying the accuracy of these SPECT for various isotopes will provide valuable reference information for the design of CZT-based pre-clinical SPECT system imaging protocols.