Mycoplasma pneumoniae causes respiratory tract infection in people of all ages and around the world. Mycoplasma infections are most often treated with macrolide antibiotics. In the last few years the number of infections with resistant M. pneumoniae strains to macrolides has increased. The first reports on the extent of the resistant strains appeared in Japan in 2000, then spread across Asia and after some time surfaced in Europe as well as in America. The resistance is associated with point mutations in the domain of V gene 23S rRNA on locations 2063, 2064, 2067 and 2617. The most frequent mutation is A2058G (E. coli numbering) which fits location 2063 in the bacterial genome of the M. pneumoniae and causes a high level of resistance. In the research we wanted to prepare real-time PCR with melting curve analysis to simultaneously proof the patient’s infection with the bacteria Mycoplasma pneumoniae and its sensitivity to macrolides. Using the real-time PCR we have tested 137 samples of isolated patients’ DNA with proven infection with M. pneumonia and 7 samples of M. pneumoniae DNA with the mutation in gene 23S rRNA, which was proven by the pyrosequencing method. Melting curve analysis clearly separated wild strain from mutation. The PCR test results were in accordance with the pyrosequencing method. However, a new strain with resistance to macrolides was not proven. Method sensitivity is 10 copies DNA M. pneumoniae/µL. We have proven 100 % test specificity.