Mice are now commonly used as models of human cardiovascular diseases and conditions, but it is challenging to measure blood flow velocity in small vessels such as coronary arteries. Accordingly, we have developed a method using a 2 mm diameter 20 MHz pulsed Doppler probe applied to the chest of an anesthetized mouse to measure left main coronary blood flow velocity noninvasively. We also found that coronary flow velocity could be increased from baseline (B) to hyperemic (H) levels by changing the concentration of isoflurane gas anesthesia from 1% to 2.5% in oxygen and that the H levels are similar to or higher than those induced by adenosine. We used the ratio H/B to estimate coronary flow reserve (CFR) in young, adult, and old mice and in mice with atherosclerosis, coronary occlusion, pressure overload, and angiotensin infusion. We found that H/B increases with age from 2.4 (young) to 3.6 (old) and is reduced by all forms of coronary and vascular disease to as low as 1.1 by pressure overload. We conclude that CFR can be measured noninvasively and serially in mice as their cardiovascular systems adapt and remodel to various imposed or natural conditions, and that left main coronary flow reserve may be a good index of global cardiac function.