Lipocalins are potential targets for drug development, and therefore it is important to understand the common and distinct features of their ligand-binding sites. Here, the sequence and structural features of ligand-binding sites in kernel and outlier lipocalins were analyzed and compared. Ligand-binding sites of kernel lipocalins were sequentially more diverse than outlier lipocalins. The ligand-binding sites of the two subgroup lipocalins were relatively hydrophobic and exhibited positive or neutral net-charges, well- correlated with the hydrophobic ligands with neutral or acidic functional groups. There was a propensity of three hydrophobic residues, Phe, Leu and Tyr in the ligand-interacting residues of kernel lipocalins, whereas outlierlipocalins showed relatively lower propensity. Finally, six and ten crucial ligand-interacting positions were identified in the kernel and outlier lipocalins, respectively. These identified features are expected to be a theoretical reference for the engineering and exploration of kernel and outlier lipocalins.