Objective To establish the method of 1H-MRS in the determination of myocardial triglyceride content (MTC) in vivo and test its reproducibility. Methods Ten healthy volunteers were recruited after routine physical examination. Cardiac images of the four-chamber and short axis views were collected. Spectroscopic volumes of interest were positioned within the interventricular septum on the end-systolic images. PRESS sequence spectroscopic data acquisition was triggered by electrocardiograph-derived R wave and synchronized with respiratory cycle at end-exhalation. MTC was calculated by the ratio of areas of resonances for lipid and water. After three months 1H-MRS of the heart were conducted again to investigate the reproducibility of the method. MTC before and after three months was compared by using paired t test. The correlation between MTC and clinical data was determined by using correlation analysis. Results MTC in healthy volunteers were (0. 46 ± 0. 17) % and (0. 48 ± 0. 19) % for the baseline and follow-up respectively. No difference was identified (t = - 0. 569, P = 0. 583). MTC was not correlated with body mass index [BMI(21.1±2. 1) kg/m2,r=0. 271,P=0.449], waist circumference [(77.9 ± 4. 7) cm,r = 0. 105, P = 0. 772], waist-to-hip ratio (WHR 0. 81 ± 0. 01, r = 0. 172, P = 0. 635), systolic blood pressure [SBP(111 ± 16) mm Hg(1 mm Hg =0. 133 kPa) ,r =0. 267,P =0. 456], diastolic blood pressure [DBP(71 ± 9) mm Hg,r = -0. 553,P =0. 097], fasting glucose [FPG(4. 77 ± 0. 44) mmol/L, r =0. 568, P =0. 087], total cholesterol [TC (4. 25 ± 0. 55) mmol/L, r = 0. 067, P = 0. 854] and triglyceride [TG (1.07±0.48) mmol/L,r =0.478,P = 0. 162]. Conclusions Myocardial triglyceride content can be quantitatively determined by 1H-MRS. The reproducibility of myocardial 1H-MRS was good for clinical purpose.