Mycoplasmopsis bovis is a significant pathogen causing substantial economic losses in cattle, yet its within-herd microevolution remains poorly understood. This study aimed to characterize phenotypic and genomic variations within a dominant ST-52 clone circulating in a closed dairy herd. We isolated M. bovis from respiratory (n = 11) and milk (n = 5) samples. Phenotypic characterization included biofilm formation, antimicrobial susceptibility testing, and cellular invasion assays. Whole-genome sequencing was performed on four representative isolates to identify genetic variations. All isolates were genetically identical according to MLST (ST-52). However, significant phenotypic diversity was observed. Biofilm formation capacity varied significantly (OD595 from 0.25 to 1.10), and resistance to doxycycline was higher in nose swabs (100%) than milk isolates (20%). Cellular invasion assays demonstrated that all isolates could invade bovine-derived cells (MDBK, MAC-T, EBL, and PBMC), but the invasion efficiency differed by strain and cell type. These findings confirm the circulation of a single genetic lineage within a closed herd while highlighting significant phenotypic diversification in biofilm formation, antibiotic resistance, and cellular invasiveness. The results provide evidence consistent with microevolution and underscore the adaptive potential of M. bovis. This study underscores the adaptive potential of M. bovis during within-host colonization and cross-tissue transmission, providing critical insights for optimizing herd management and treatment strategies.