Although previous studies have confirmed that 23S rRNA gene mutation could be responsible for most of macrolide resistance in M. catarrhalis, a recent study suggested otherwise. Next generation sequence based comparative genomics has revolutionized the mining of potential novel drug resistant mechanisms. In this study, two pairs of resistant and susceptible M. catarrhalis isolates with different multilocus sequence types, were investigated for potential differential genes or informative single nucleotide polymorphisms (SNPs). The identified genes and SNPs were evaluated in 188 clinical isolates. From initially 12 selected differential genes and 12 informative SNPs, 10 differential genes (mboIA, mcbC, mcbI, mboIB, MCR-1794, MCR-1795, lgt2B/C, dpnI, mcbB, and mcbA) and 6 SNPs (C619T of rumA, T140C of rplF, G643A of MCR-0020, T270G of MCR-1465, C1348A of copB, and G238A of rrmA) were identified as possibly linked to macrolide resistance in M. catarrhalis. Most of the identified differential genes and SNPs are related to methylation of ribosomal RNA (rRNA) or DNA, especially MCR-0020 and rrmA. Further studies are needed to determine the function and/or evolution process, of the identified genes or SNPs, to establish whether some novel or combined mechanisms are truly involved in M. catarrhalis macrolide resistance mechanism.