Diagnosis of Mycoplasma pneumoniae infection is challenging due to the fastidious nature of the pathogen, the considerable seroprevalence, and the possibility of transient asymptomatic carriage. During recent years, various new techniques have been adapted for the diagnosis of M. pneumoniae infection, notably in the field of molecular biology. Standard polymerase chain reaction (PCR) is currently the method of choice for direct pathogen detection, but several PCR-related methods provide enhanced sensitivity or more convenient handling procedures, and have been successfully applied for research purposes. Among these techniques are real-time PCR, nested PCR, reverse transcriptase PCR (RT-PCR) and multiplex PCR. Generally, amplification-based methods have replaced hybridization assays and direct antigen detection. Serology, which is the basic strategy for mycoplasma diagnosis in routine clinical practice, has been improved by the widespread availability of sensitive assays for separate detection of different antibody classes. For the diagnosis of mycoplasma pneumonia, serology and direct pathogen detection should be combined. Extrapulmonary diseases may be diagnosed by direct pathogen detection alone, but the value of this diagnostic approach is limited by the probably immunologically mediated pathogenesis of some manifestations. This review summarizes the current state of Mycoplasma pneumoniae diagnosis, with special reference to molecular techniques. The value of different methods for routine diagnosis and research purposes is discussed.