Previous studies indicate that macrolide resistance in Moraxella catarrhalis isolates is less common in adults than in children. However, few studies have investigated M. catarrhalis macrolide resistance mechanisms in adult patients. In this study, 124 M. catarrhalis isolates were collected from adult patients in a Chinese tertiary hospital, between 2010 and 2013, and investigated for antimicrobial resistance. We found that only seven isolates were macrolide resistant and all exhibited high-level macrolide resistance (minimum inhibitory concentrations >256 μg/ml). Multilocus sequence typing (MLST) suggested that M. catarrhalis has a diverse population; in particular, both pulsed-field gel electrophoresis and MLST revealed that all the seven high-level macrolide-resistant M. catarrhalis belonged to different clones. A 934-bp 23S rRNA gene sequencing showed that only nine isolates (including all the seven macrolide-resistant isolates) had mutations within the studied region, and only the seven macrolide-resistant isolates had mutation of A2330T. No other known macrolide-resistance determinant genes (ermA, ermB, mefA, or mefE) were detected. These findings support previous studies in children on M. catarrhalis macrolide-resistant isolates and suggest that the 23S rRNA gene A2330T mutation is responsible for the high M. catarrhalis macrolide resistance. The findings prompted us to successfully develop a simple allele-specific polymerase chain reaction assay for high-level macrolide-resistant 23S rRNA gene A2330T mutation for future clinical and further surveillance use.