Mycoplasma spp. are extracellular bacteria that colonize on the respiratory epithelium of humans and animals. It is a causative agent of pneumonia commonly complicated by opportunistic infectious bacteria. Mycoplasma spp. infection cause relatively mild disease in the absence of environmental stressors, but when complicated by secondary bacterial invaders the resultant disease can cause obvious clinical disease and severe production losses in intensively reared pigs Mycoplasma spp. are highly fastidious bacteria, difficult to culture and slow growing. Many species of Mycoplasma spp. are important pathogens causing respiratory infection in animals and known to induce huge economic losses. The aims of the present study were to develop a rapid isolation and culture method of wild type Mycoplasma spp. in pigs. We used Mycoplasma spp. genus specific direct PCR without DNA extraction procedure using PhireⓇ Animal Tissue Direct PCR Kit from the lung tissues with pneumonia lesions. Therefore, we could save the time for tissue processing and increase the accuracy of Mycoplasma spp. inclusion prediction in lung tissues. Thereafter, we used the optimized media to isolate and culture Mycoplasma spp. As the results, Mycoplasma spp. could be isolated and cultured quickly and efficiently. These results could provide an efficient strategy and method for the rapid and accurate isolation and culture of wild type Mycoplasma spp. in pigs.