The classical human pancreatic lipase (HPL), the guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the phospholipase A1 from hornet venom (DolmI PLA1) illustrate three interesting steps in the molecular evolution of the pancreatic lipase gene family towards different substrate selectivities. Based on the known 3D structures of HPL and a GPLRP2 chimera, as well as the modeling of DolmI PLA1, we review here the structural features and the kinetic properties of these three enzymes for a better understanding of their structure-function relationships. HPL displays significant activity only on triglycerides, whereas GPLRP2 displays high phospholipase and galactolipase activities, together with a comparable lipase activity. GPLRP2 shows high structural homology with HPL with the exception of the lid domain which is made of five amino acid residues (mini-lid) instead of 23 in HPL. The lid domain deletion in GPLRP2 allows the free access to the active site and reduces the steric hindrance towards large substrates, such as galactolipids. The role of the lid domain in substrate selectivity has been investigated by site-directed mutagenesis and the substitution of HPL and GPLRP2 lid domains. The addition of a large-size lid domain in GPLRP2 increases the substrate selectivity for triglycerides by depressing the phospholipase activity. The phospholipase activity is, however, not induced in the case of the HPL mutant with GPLRP2 mini-lid. Therefore, the presence of a full-length lid domain is not the unique structural feature explaining the absence of phospholipase activity in HPL. The 3D structure of the GPLRP2 chimera and the model of DolmI PLA1 reveal a higher hydrophilic/lipophilic balance (HLB) of the surface loops (beta5 loop, beta9 loop, lid domain) surrounding the active site, as compared to the homologous loops in HPL. This observation provides a potential explanation for the ability of GPLRP2 and DolmI PLA1 to hydrolyze polar lipids, such as phospholipids. In conclusion, the beta5 loop, the beta9 loop, and the lid domain play an essential role in substrate selectivity towards triglycerides, phospholipids and galactolipids.