Current antibiotic resistance studies often focus on individual protein variants, neglecting broader protein family dynamics. Dihydrofolate reductase (DHFR), a key antibiotic target, has been extensively studied using deep mutational scanning, yet resistance mechanisms across this diverse protein family remain poorly understood. Using DropSynth, a scalable gene synthesis platform, we designed a library of 1,536 synthetic DHFR homologs representing 778 species of bacteria, archaea, and viruses, including clinically relevant pathogens. A multiplexed