β-lactamases are enzymes which are commonly produced by bacteria and which degrade the β-lactam ring of β-lactam antibiotics, namely penicillins, cephalosporins, carbapenems, and monobactams, and inactivate these antibiotics. We performed a rational and comprehensive investigation of β-lactamases in different biological databases. In this study, we constructed hidden Markov model profiles as well as the ancestral sequence of four classes of β-lactamases (A, B, C, and D), which were used to identify potential β-lactamases from environmental metagenomic (1206), human microbiome metagenomic (6417), human microbiome reference genome (1310), and NCBI's nonredundant databases (44101). Our analysis revealed the existence of putative β-lactamases in the metagenomic databases, which appeared to be similar to the four different molecular classes (A-D). This is the first report on the large-scale phylogenetic diversity of new members of β-lactamases, and our results revealed that metagenomic database dark-matter contains β-lactamase-like antibiotic resistance genes.