Objective: Simultaneous acquisition of 99mTc and 123I was evaluated using a preclinical SPECT scanner with cadmium zinc telluride (CZT)-based detectors. Methods: 10-ml cylindrical syringes contained about 37 MBq 99mTc-tetrofosmin (99mTc-TF) or 37 MBq 123I-15-(p-iodophenyl)-3R,S-methyl pentadecanoic acid (123I-BMIPP) were used to assess the relationship between these SPECT radioactive counts and radioactivity. Two 10-ml syringes contained 100 or 300 MBq 99mTc-TF and 100 MBq 123I-BMIPP to assess the influence of 99mTc upscatter and 123I downscatter, respectively. A rat-sized cylindrical phantom also contained both 100 or 300 MBq 99mTc-TF and 100 MBq 123I-BMIPP. The two 10-ml syringes and phantom were scanned using a pinhole collimator for rats. Myocardial infarction model rats were examined using 300 MBq 99mTc-TF and 100 MBq 123I-BMIPP. Two 1-ml syringes contained 105 MBq 99mTc-labeled hexamethylpropyleneamine oxime (99mTc-HMPAO) and 35 MBq 123I-labeled N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) nortropane (123I-FP-CIT). The two 1-ml syringes were scanned using a pinhole collimator for mice. Normal mice were examined using 105 MBq 99mTc-HMPAO and 35 MBq 123I-FP-CIT. Results: The relationship between SPECT radioactive counts and radioactivity was excellent. Downscatter contamination of 123I-BMIPP exhibited fewer radioactive counts for 300 MBq 99mTc-TF without scatter correction (SC) in 125–150 keV. There was no upscatter contamination of 99mTc-TF in 150–175 keV. In the rat-sized phantom, the radioactive count ratio decreased to 4.0 % for 300 MBq 99mTc-TF without SC in 125–150 keV. In the rats, myocardial images and radioactive counts of 99mTc-TF with the dual tracer were identical to those of the 99mTc-TF single injection. Downscatter contamination of 123I-FP-CIT was 4.2 % without SC in 125–150 keV. In the first injection of 99mTc-HMPAO and second injection of 123I-FP-CIT, brain images and radioactive counts of 99mTc-HMPAO with the dual tracer in normal mice also were the similar to those of the 99mTc-HMPAO single injection. In the first injection of 123I-FP-CIT and second injection of 99mTc-HMPAO, the brain images and radioactive counts with the dual tracer were not much different from those of the 123I-FP-CIT single injection. Conclusions: Dual-tracer imaging of 99mTc- and 123I-labeled radiotracers is feasible in a preclinical SPECT scanner with CZT detector. When higher radioactivity of 99mTc-labeled radiotracers relative to 123I-labeled radiotracers is applied, correction methods are not necessarily required for the quantification of 99mTc- and 123I-labeled radiotracers when using a preclinical SPECT scanner with CZT detector.