Genetically modified mouse models of many human diseases reflecting cardiovascular alterations are currently available. To date, little information on absolute myocardial perfusion in mice is found in the literature. High‐resolution quantitative myocardial blood flow maps (in‐plane resolution 156 × 312 μm², slice thickness 1.5 mm) have been obtained noninvasively within 25 min at 4.7 T in 30 freely breathing C57/Bl6J mice using electrocardiogram‐ and respiration‐gated spin labeling magnetic resonance imaging (MRI). Regional myocardial blood flow measurements were carried out, and the effects of isoflurane at two different concentrations and ketamine/xylazine anesthesia were assessed. The mean blood flow value in the left ventricular myocardium was 6.0 ± 1.9 mL g⁻¹ min⁻¹ under ketamine/xylazine and 6.9 ± 1.7 mL g⁻¹ min⁻¹ (group average ± SD) under isoflurane (1.25%). Under the influence of higher isoflurane concentration (2.00%), myocardial blood flow increased dramatically to 16.9 ± 1.8 mL g⁻¹min⁻¹ with no significant change in heart rate. This work illustrates the feasibility of noninvasive quantitative myocardial perfusion mapping in mice using MRI. The study of the influence of anesthesia shows that myocardial blood flow is highly sensitive to isoflurane concentration. The method employed offers a noninvasive approach to longitudinal studies of murine models of cardiac disease. Magn Reson Med 53:601–606, 2005.