Background <p>Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is a common bacterial pathogen responsible for hospital- and community-acquired infections and typically exhibits a β-hemolytic phenotype. Notably, MRSA with an incomplete hemolytic phenotype (SIHP-MRSA) has been reported in few studies and has been shown to display altered virulence and resistance to last-resort antibiotics. However, the detailed genotypic and phenotypic characteristics of SIHP-MRSA clinical isolates remain unclear. This study aims to analyze the genotypic and phenotypic characteristics of SIHP-MRSA clinical isolates using a large sample size and to explore potential molecular mechanisms underlying the incomplete hemolytic phenotype, altered biofilm production capability, and elevated antimicrobial resistance of SIHP-MRSA.</p> Methods <p>MRSA clinical isolates were obtained from patients admitted to the Affiliated Hospital of Inner Mongolia Medical University during 2010–2024, and hemolytic patterns were visually assessed on blood agar plates. Multilocus sequence typing (MLST), staphylococcal protein A (spa) typing, and staphylococcal cassette chromosome mec (<i>SCCmec</i>) typing were used to characterize the molecular genotypes of SIHP-MRSA isolates, whereas PCR was used to identify the distribution of virulence genes. Mutations in the <i>agrA</i> gene among SIHP-MRSA isolates were identified via Sanger sequencing. Biofilm formation capabilities were assessed using Congo red agar and crystal violet staining, while antimicrobial resistance profiles were determined using the VITEK-2 Compact system and the broth microdilution method.</p> Results <p>Among 1,122 MRSA clinical isolates analyzed in this study, 28.5% (320/1,122) were classified as SIHP-MRSA. The majority of isolates were isolated from lower respiratory tract samples (61.8%), followed by whole blood (9.0%) and secretions (8.5%). Molecular typing results indicated that ST5-t2460-II (85.6%, 274/320) and ST59-t437-IVa (10.3%, 33/320) were the dominant clones. Among these isolates, 21.5% (69/320) carried the <i>pvl</i> gene and 56.8% (182/320) carried <i>tsst-1</i>. Notably, 35 isolates carried both <i>pvl</i> and <i>tsst-1</i> genes, primarily belonging to ST5-t2460-II (93.5%, 29/31). Overall, 13.4% (43/320), 45.9% (147/320), and 40.6% (130/320) of SIHP-MRSA isolates were classified as weak, moderate, and strong biofilm producers, respectively. The <i>agrA</i>-I238K mutation occurred in 89.7% (287/320) of isolates, and 57.1% (164/287) and 61.3% (176/287) of isolates exhibited reduced susceptibility to teicoplanin and daptomycin, respectively. Elevated teicoplanin MICs (≥ 2&#xa0;µg/mL) and daptomycin MICs (≥ 1&#xa0;µg/mL) were significantly more frequent in SIHP-MRSA isolates with the <i>agrA</i>-I238K mutation than in isolates with a complete hemolytic phenotype (SCHP-MSRA) (<i>P</i> = 0.007).</p> Conclusions <p>This study is the first to report a high prevalence of SIHP-MRSA in a tertiary teaching hospital in China and to describe its genotypic diversity. These isolates showed enhanced biofilm formation, reduced susceptibility to daptomycin and teicoplanin, strong nosocomial transmission potential, and higher carriage rates of <i>pvl</i> and <i>tsst-1</i> virulence genes. The significant genotypic and phenotypic features of SIHP-MRSA identified in this study highlight the previously neglected clinical significance of this MRSA subtype and emphasize the need for further exploration into its pathogenicity among vulnerable populations and its underlying molecular mechanisms.</p>