Abstract: First of all, the conserved genes LytA of SP, P1 of MP and ompA of CP were chosen as targets for design of primers and TaqMan probes. The Multiplex RT-PCR methods were developed for diagnosis of three pathogens. Secondly, according to sequence ofcps, the TaqMan/MGB probes were designed and the Multiplex RT-PCR methods were developed for serotyping of SP. Last, the ermB, mefA were chosen as targets for design of primers and probes for SP drug resistance, then it used for the detection of drug resistance in CAP clinical samples. The standard curve of multiplex RT-PCR showed great linear relationship. The sensitivity of methods for three pathogens were 10² copies/mL, while the detection range were 10²~10⁷ copies/mL and no cross reaction with other microbial floras. 304 pathogen-positive clinical specimens was identified from 512 sputum, the positive of SP, MP and CP were 189(62.17%), 83(27.30%)and 32(10.53%),respectively. There were good consistency between Multiplex RT-PCR and Simplex RT-PCR, and the results between Multiplex RT-PCR and Sequence methods were no significant difference. The serotype of SPin 189 positive samples were 19 (19.58%, 37/189), 23 (15.87%, 30/189), 6 (13.23%, 25/189), 14 (6.3%, 12/189), other serotypes (22.75%, 43/189), and unknown serotypes (22.22%, 42/189). The gene mutations of SP resistance strains were identified as follows:ermB (60.85%, 115/189), mefA (29.63%, 56/189), and both ermB and mefA (7.94%, 15/189).