The goal is to develop and evaluate two methods to extract interventricular sulcus (IS) from 4D cardiac gated (CG) myocardial perfusion (MP) PET images for use in quantitative cardiac motion estimation. In Method 1, the entire myocardium was first segmented from the 3D MP PET image of each CG frame by 3D region growing. The left ventricle (LV) was then extracted by 3D image erosion and dilation, and the right ventricle (RV) by subtracting the LV from the segmented myocardium. Finally, the IS was identified from the overlap of the extracted LV and RV. In Method 2, the inner boundary (mB) of the RV was first extracted by segmenting the blood pool (BP) within the RV using 3D region growing. The septal side of the BP boundary, or the LV outer boundary (OutB) within the RV, was separated out. We then identified segments of the LV OutB on the anterior (Ant) and posterior (Post) sides of the RV. The three extracted line segments were fitted with a B-spline curve while the lateral side of the RV mB was extrapolated using B-spline. The intersection points of the fitted curves were identified as the Ant and Post IS which, when assembled from all short-axis images, formed entire IS. The two IS extraction methods were applied to realistic CG MP PET images simulated from the 4D XCAT phantom at PET system resolution from 0.6mm to 4.5mm for 4 CG frames. The accuracy of the extracted IS were compared with the true IS from the XCAT. For both methods, the errors of the extracted IS locations increased with poorer PET system resolution with the Ant IS showing lower accuracy than the Post IS. Method 1 achieved lower accuracy than Method 2 and failed to provide reliable estimates at 4.5mm system resolution. We conclude the B-spline based interpolation and extrapolation curve fitting method was capable of extracting the IS with high reliability and accuracy from the 4D CG MP PET images obtained from state-of-the-art and PET systems and will be useful in the cardiac motion estimation.