The axon initial segment (AIS) is a highly specialized axonal compartment where the action potential is initiated. The heterogeneity of AISs has been suggested to occur between interneurons and pyramidal neurons (PyNs), which likely contributes to their unique spiking properties. However, whether the various characteristics of AISs can be linked to specific PyN subtypes remains unknown. Here, we report that in the prelimbic cortex (PL) of the mouse, two types of PyNs with axon projections either to the contralateral PL or to the ipsilateral basal lateral amygdala, possess distinct AIS properties reflected by morphology, ion channel expression, action potential initiation, and axo-axonic synaptic inputs from chandelier cells. Furthermore, projection-specific AIS diversity is more prominent in the superficial layer than in the deep layer. Thus, our study reveals the cortical layer- and axon projection-specific heterogeneity of PyN AISs, which may endow the spiking of various PyN types with exquisite modulation.