Radiofrequency catheter ablation (RFA) targets the left atrial appendage isthmus (LAA isthmus) and mitral isthmus for treatment of atrial fibrillation. However, proximity of left circumflex artery (LCxA) and great cardiac vein (GCV) in the isthmuses poses fatal risks during ablation. This study investigated relationships of LCxA and GCV across three lines in the LAA and mitral isthmus, using 15 human cadaveric hearts. Distances between the vessels and the endocardium, myocardium, and perivascular fat thickness were measured. The results showed that LCxA was mostly consistently located in lower atrial segments and GCV was in lower/upper atrial segments, with change of course mainly observed in the middle of the LAA. The LCxA was found as close as 3-5 mm from the lower border of the LAA isthmus in 80% of specimens, at a depth of 2-3 mm within the LAA isthmus, where 1 mm consisted of myocardium and the remainder was fat, which may not provide adequate protection due to the possibility of liquefaction of fat with heat application. The effective myocardial thickness was consistently 1 mm across all cases in both isthmuses. LCxA was 2 mm in second and third sections of LAA isthmus ("careful segment"). LCxA distances from left inferior pulmonary vein opening was 5 to 12 mm, occasionally dangerously close as <1 mm in 16% of cases. This study measured LCxA and GCV in the LAA and mitral isthmus across three lines for the first time in the Indian population, aiding surgeons in RFA planning.