Metagenomic-based studies have predicted an extraordinary number of potential antibiotic-resistant genes (ARGs). These ARGs are hidden in various environmental bacteria and may become a latent crisis for antibiotic therapy via horizontal gene transfer. In this study, we focus on a resistance gene cph, which encodes a phosphotransferase (Cph) that confers resistance to the antituberculosis drug capreomycin (CMN). Sequence Similarity Network analysis classified 353 Cph homologues into five major clusters. We examined the function and targeted antibiotics of three putative resistance proteins in cluster I via bioinformatics, biochemical, and protein structural analysis. We found that the proteins in cluster I are from various species and indeed confer resistance to CMN. This study contributes towards understanding the sequence-structure-function relationships of the phosphorylation resistance genes that confer resistance to CMN.