Serial imaging with micro SPECT/CT is an important 3D in vivo technique used to study heart disease in small‐animal models and develop new therapies and new radiotracers. Measurement of myocardial perfusion uniformity (PH), ejection fraction (EF), end systolic volume (ESV) and, end diastolic volume (EDV) in rat model are important indices of heart function that can be derived from SPECT images. Knowledge of the uncertainty of these measurements is critical to discerning true changes and to determining sample sizes. The aim of this study is to produces the inter‐ and intra‐ subject reproducibility of left ventricular volumes, ejection fraction and perfusion homogeneity with Tl‐201, a common cardiac perfusion tracer, in a rat model using micro SPECT/CT. The scanner has four heads with nine pinholes each (total 36 pinholes). Three healthy rats were injected with 0.5mCi of Tl‐201 and scanned weekly for five weeks. Each scan lasted 30 min and began 30 min post‐injection. The images were reconstructed and processed with a clinical software package (4DM‐SPECT) to get the heart functions (EDV, ESV, EF and PH). EDV and ESV were corrected for changes in rat weight. The standard deviation in the measured values (across scans) was 5.88% for EDV and 6.90% for ESV, 3.2% for EF, and 6% for PH. The reproducibility between rats was similar.