Background: Mycoplasma pneumoniae is one of the most common causes of atypical pneumonia, which is almost asymptomatic and self-limited. Objectives: The current study aimed at investigating the prevalence of M. pneumoniae among children with pneumonia in Ahvaz, Iran, using bacterial culture growth, polymerase chain reaction (PCR), and serology tests. Methods: A total of 136 throat swab and serum specimens were collected from patients with pneumonia. The specimens were cultured on pleuropneumonia-like organisms (PPLO) agar. Molecular identification of the throat swab specimens was performed using the amplification of P1 gene. Determination of M. pneumoniae-specific antibodies (IgG and IgM) in the sera was carried out by the enzyme-linked immunosorbent assay (ELISA) technique. Results: In the current study, the acute infection was detected in 16 cases. Moreover, 3 out of 136 cases had positive results in their bacterial culture. Mycoplasma pneumoniae DNA was detected in 11 of the 136 cases. An acceptable titer of IgM was observed in 12 cases. On the other hand, 4-fold or greater titer of IgG was detected in 14 cases. Conclusions:The results of the current study suggested that the combination of PCR and the serology results were effective to detect M. pneumoniae. Moreover, the combination of PCR and IgM results can detect all cases of acute infection with M. pneumoniae in children.