The article provides an updated functional classification of β-lactamases, which are enzymes that hydrolyze β-lactam antibiotics, leading to resistance in Gram-negative bacteria. The classification is based on the 1995 proposal by Bush et al., incorporating substrate and inhibitor profiles to correlate enzyme properties with clinical isolates. The major groups include Group 1 (class C cephalosporinases), Group 2 (classes A and D broad-spectrum, inhibitor-resistant, and extended-spectrum β-lactamases), and Group 3 (metallo-β-lactamases). New subgroups within these groups are described, based on specific attributes of individual enzymes. The classification aims to provide a systematic approach to tracking these enzymes, which are crucial for selecting appropriate therapy in clinical settings. The article also discusses the diversity of β-lactamases, their evolution, and the challenges in their classification, emphasizing the need for a comprehensive set of criteria for describing new β-lactamases.The article provides an updated functional classification of β-lactamases, which are enzymes that hydrolyze β-lactam antibiotics, leading to resistance in Gram-negative bacteria. The classification is based on the 1995 proposal by Bush et al., incorporating substrate and inhibitor profiles to correlate enzyme properties with clinical isolates. The major groups include Group 1 (class C cephalosporinases), Group 2 (classes A and D broad-spectrum, inhibitor-resistant, and extended-spectrum β-lactamases), and Group 3 (metallo-β-lactamases). New subgroups within these groups are described, based on specific attributes of individual enzymes. The classification aims to provide a systematic approach to tracking these enzymes, which are crucial for selecting appropriate therapy in clinical settings. The article also discusses the diversity of β-lactamases, their evolution, and the challenges in their classification, emphasizing the need for a comprehensive set of criteria for describing new β-lactamases.