Abstract During the winter 2023–2024, an upsurge of Mycoplasma pneumoniae (M.pneumoniae) was noted in the Netherlands. To investigate the distribution of M.pneumoniae sequence types from different patient populations and to explore genotypic macrolide resistance which is common in East Asia but not (yet) in Europe. M.pneumoniae positive throat/nasal samples from participatory respiratory surveillance, patients visiting general practitioners with an acute respiratory infection including community acquired pneumonia (CAP) and hospitalised patients with CAP were included, representing different disease severity. The M.pneumoniae were typed with multilocus sequence typing and the 23 S rRNA region was sequenced to determine macrolide resistance markers. In total, 153 M.pneumoniae were sequenced, six sequence types (STs) and only one bacterium with macrolide resistance marker were detected. No link between STs or bacterial load (PCR cycle threshold) and source population of M.pneumoniae was detected. In the Netherlands, the M.pneumoniae upsurge in 2023–2024 existed of multiple commonly found STs. No link between ST and severity of illness was detected. Macrolide resistance remained sporadic.