An important role in the treatment regimens for Mycoplasma pneumoniae infections is played by macrolide (ML) antibiotics. In the past few years, however, a steady increase has been detected in the worldwide prevalence of ML-resistant (ML(r)) M. pneumoniae strains. It is obvious that this increase necessitates a continuous monitoring of ML(r) and, when detected, modification of antibiotic treatment modalities. Previously, we developed a pyrosequencing-based assay system for the genetic determination of ML(r) as well as molecular typing of M. pneumoniae. In this study, the sensitivity of this system was improved by the inclusion of a nested-PCR protocol. The modified system was applied to 114 M. pneumoniae-positive specimens that were obtained from a collection of 4,390 samples from patients with acute respiratory tract infections. These samples were collected between 1997 and 2008 in The Netherlands. The pyrosequencing system produced reliable data in 86% of the specimens that contained >500 M. pneumoniae genome copies/ml of patient sample. Each of these samples contained DNA of the ML-sensitive genotype. While 43% of the samples were found to harbor the M. pneumoniae subtype 1 genotype, 57% contained the subtype 2 genotype. We conclude that the pyrosequencing-based assay system is a useful tool for ML(r) determination and molecular typing of M. pneumoniae in patient samples. ML(r)-associated M. pneumoniae genotypes, however, were not found in the current study population.