Bacterial resistance to antibiotics is a serious medical and public health concern worldwide. Such resistance is conferred by a variety of mechanisms, but the extensive variability in levels of resistance across bacteria is a common finding. Understanding the underlying evolutionary processes governing this functional variation in antibiotic resistance is important as it may allow the development of appropriate strategies to improve treatment options for bacterial infections. The main objective of this study was to examine the functional evolution of β-lactamases, a common mechanism of enzymatic resistance that inactivates a widely used class of antibiotics. We first obtained β-lactamase protein sequences and minimal inhibitory concentration (MIC), a measure of antibiotic function, from previously published literature. We then used a molecular phylogenetic framework to examine the evolution of β-lactamase functional activity. We found that the functional activity of antibiotic resistance mediated by β-lactamase has evolved in a convergent manner within molecular classes, but is not associated with any single amino acid substitution. This suggests that the dynamics of convergent evolution in this system can vary between the functional and molecular (sequence) levels. Such disassociation may hamper bioinformatic approaches to antibiotic resistance determination and underscore the need for (less efficient but more effective) activity assays as an essential step in evaluating resistance in a given case.