[Background] Anatomical variations and adjacent structures of left atrium can cause procedural difficulties as well as complications in creating additional linear lesions during atrial fibrillation (AF) ablation. [Methods] Multi-detector computed tomography (MDCT) data from 140 consecutive patients (40 with AF, 84 males, 59짹11 years old) and additional 10 heart specimens were analyzed for their anatomical characteristics at three types of mitral isthmus lines: anteromedial (AM), anterolateral (AL), and posterolateral (PL) lines (from right superior, left superior, and left inferior pulmonary veins to 10, 12, and 4 o���clock position of the mitral annulus, respectively). [Results] The length was shortest at the PL lines: 46.7짹7.6, 43.9짹6.2 and 31.4짹6.1mm at the AM, AL, and PL line in MDCT, respectively; 54짹7, 47짹16, and 31짹6 mm in specimens, respectively; p<0.05 for PL vs. AM or vs. AL line both in MDCT and specimens. The maximal thickness of myocardium was greatest at the AL lines: 2.1짹0.7, 3.2짹1.0, 2.4짹0.8mm in MDCT; 4.3짹0.8, 5.0짹0.9, 3.9짹1.1mm in specimens; p<0.05 for AL vs. AM and vs. PL line in MDCT; p=0.114 and 0.035 in specimens, respectively. Ridge, cord-like structure or diverticulum was found most frequently at the AM lines (20%, 0%, 11.4% in MDCT; 20%, 0%, 0% in specimens). Sinus nodal artery (SNA) was almost always found near the AM (MDCT, 100%; specimens, 90%) even when it originated from the right coronary artery (54% in MDCT). SNA was also found near the AL lines (MDCT, 46.3%; specimens, 30%). The left coronary artery and cardiac vein were closest to the PL lines. The trend of these findings was not significantly altered with the presence of AF. [Conclusions] Among the three mitral isthmus lines, the PL lines were shortest, but closest to the left coronary arteries. Myocardium was thickest at the AL line, and SNAs were frequently found on the AM and AL lines. In addition, the AM and PL lines had more obstacles at their locations than the AL lines. Among the three mitral isthmus lines, none was superior to the others in every aspect, thus pre-ablation CT images can be helpful in planning the ablation strategy.Further studies with clinical outcomes after catheter ablation are required to clarify the clinical impact of these anatomic findings.