This study aimed to quantitatively evaluate population variability, regional uniformity and repeatability of myocardial blood flow measurements using [13N]-ammonia and small animal PET. Serial PET scans were conducted on Sprague-Dawley rats using [13N]-ammonia to study relative perfusion and absolute myocardial blood flow (ml/min/g). FlowQuant automated analysis software was used to produce five-segment polar maps to investigate regional myocardial blood flow differences. The interobserver and intraobserver repeatability was assessed quantitatively using Bland-Altman analysis. Absolute myocardial blood flow values were 4.3 ± 1.1 ml/min/g, corresponding to a population variability of 25.5%. There were significant age-related increases in resting myocardial blood flow (r2=0.59, P<0.001). The test-retest differences had a coefficient of repeatability of 24.5% of the mean myocardial blood flow. The operator variability was small, relative to the population variability. Repeatable myocardial blood flow values are minimally influenced by operator intervention. However, age-related myocardial blood flow increases must be taken into account when comparing measurements between experimental groups.