Mycoplasma pneumoniae is increasingly recognized as a common and an important pathogen in community-acquired respiratory tract infections (RTIs) and pneumonia, particularly in school-age children and young adults. To determine the incidence and antibiotic susceptibility of M. pneumoniae at the main hospitals in Riyadh, Saudi Arabia, we studied 552 respiratory samples from patients diagnosed with different clinical diagnosis. The isolation, identification, enumeration and antibiotic resistance test for M. pneumoniae were performed using Pneumofast kit. There were 194 patients (35.1%) with current M. pneumoniae infection, mostly among younger age groups, with pneumonia the most common underlying clinical condition. All tested isolates were susceptible to four antibiotics included in the Pneumofast kit, doxycycline, minocycline, ciprofloxacine and erythromycin. The findings suggest that M. pneumonia infection in Saudi Arabia is more common among younger age groups, and pneumonia is the most common underlying clinical condition among patients with M. pneumoniae infection, that cannot be distinguished from other respiratory infections on the basis of clinical and radiographic diagnosis alone.