Abstract: Nuclear cardiology plays an important role in management of cardiac patients with known or suspected coronary artery disease (CAD). CAD is the leading cause of premature death and permanent disability. Myocardial perfusion imaging (MPI) with radiotracers is a non-invasive technique that shows the heart function and the areas of perfusion defects. In order to evaluate perfusion defects accurately, the radiotracer used in MPI studies must be taken up into myocardium in proportion to the regional myocardial blood flow. At present, ^99mTc-TEBO is commonly used in clinic for rapid myocardial perfusion imaging, which has high initial uptake but poor myocardial retention, so it is necessary to optimize its structure. In this study, a group of novel ^99mTc-labeled rapid boric acid derivatives tracers for myocardial perfusion imaging, including ^99mTc-2SP, ^99mTc-4LPA and ^99mTc-2MDM were prepared in this study. The preparation time of these tracers was 30 min, and all of them were colorless clear liquid. The radiochemical purity of the three tracers was > 95%, respectively. The above tracer agents were used to perform SPECT dynamic imaging in healthy miniature pigs and compared with ^99mTc-TEBO. After ^99mTc-TEBO injection, rapid uptake was shown in left ventricular myocardium within 0.5 min, but the myocardial radioactivity decreased rapidly to about 75% of the peak value and the ratio of the radioactivity between myocardium/blood pool was 1.63 at 10 min. Compared with ^99mTc-TEBO, ^99mTc-2SP had prolonged retention and slower elution in myocardium within 15 min after the injection when the 2-methylsulfonyl pyridine-5-boric acid group was introduced into the ^99mTc-2SP structure. The myocardial radioactivity still maintained 95% of the peak value, and the ratio of radioactivity between myocardium/blood pool was 3.75, which was significantly higher than that of ^99mTc-TEBO. However, the other two imaging agents ^99mTc-4LPA and ^99mTc-2MDM showed poor uptake in myocardium within 15 min after injection. In conclusion, ^99mTc-2SP was an ideal boric acid derivatives tracers for myocardial perfusion imaging and has prospect for clinical application in future.