Beta-lactamase (EC 3.5.2.6) synthesis, particularly in Gram-negative bacilli, is a major mechanism of natural and acquired resistance to beta-lactams, sometimes accompanied by impermeability and/or active efflux. These enzymes have been classified into four molecular classes (A–D). The serine enzymes of class A, which may be encoded by the bacterial chromosome or transferable elements and are susceptible to clinically available inhibitors (clavulanic acid, sulbactam, tazobactam, avibactam), are prevalent considering other molecular classes (B,C,D). The continual rapid development of genomic approaches and tremendous progress in automatic sequencer technology have resulted in the accumulation of massive amounts of data. A structure-based classification of class A beta-lactamases based on specific conserved motifs involved in catalytic mechanisms and/or substrate binding (S⁷⁰XXK, S¹³⁰DN, K²³⁴TG), together with E166 (Ambler numbering) and at least 24 other amino-acid residues or analogs such as G45, F66, V80, L81, L91, L101, P107, A134, L138, G143, G144, G156, L169, T181, T182, P183, was validated on 700 amino-acid sequences, including 132 representative types, but mostly probable enzyme sequences, many produced by environmental bacteria. Two subclasses (A1, A2), six major clusters or groups (e.g. natural limited-spectrum beta-lactamases (LSBL), wider spectrum beta-lactamases (WSBL), and various other clusters were identified on the basis of conserved (> 90%) and specific motifs, and residues such as S⁷⁰TFKAL, S¹³⁰DNTAANL, R¹⁶⁴XEXXLN, V²³¹GDKTG for subclass A1, S⁷⁰VFKFH, S¹³⁰DNNACDI,E¹⁶⁶XXM, and V²³¹AHKTG for subclass A2, a probable disulfide bridge C77-C123 and G236, A237, G238, and R244 for the LSBL group. This great diversity of primary structures was used as the basis for a structure-based and phylogenetic classification.