Tracer kinetic modeling with dynamic positron emission tomography (PET) requires a plasma time-activity curve (PTAC) as an input function. Several image-derived input function (IDIF) methods that rely on drawing the region of interest (ROI) in large vascular structures have been proposed to overcome the problems caused by the invasive approach for obtaining the PTAC, especially for small-animal studies. However, the manual placement of ROIs for estimating IDIF is subjective and labor-intensive, making it an undesirable and unreliable process. In this paper, we propose a novel hybrid clustering method (HCM) that objectively delineates ROIs in dynamic PET images for the estimation of IDIFs, and demonstrate its application to the mouse PET studies acquired with [ (18)F]Fluoro-2-deoxy-2-D-glucose (FDG). We begin our HCM using k-means clustering for background removal. We then model the time-activity curves using polynomial regression mixture models in curve clustering for heart structure detection. The hierarchical clustering is finally applied for ROI refinements. The HCM achieved accurate ROI delineation in both computer simulations and experimental mouse studies. In the mouse studies, the predicted IDIF had a high correlation with the gold standard, the PTAC derived from the invasive blood samples. The results indicate that the proposed HCM has a great potential in ROI delineation for automatic estimation of IDIF in dynamic FDG-PET studies.