Mycoplasma gallisepticum causes respiratory disease and production losses in poultry. Vaccination of poultry with M. gallisepticum live vaccines is an approach to reduce susceptibility to infection and to prevent the economic losses. The development and evaluation of live vaccines usually requires the involvement of several vaccine and challenge strains in the same experimental setup. Our goal was to develop a tool to allow the differentiation between a set of known M. gallisepticum strains in a quantitative manner. We developed 5 real-time PCR assays that absolutely differentiated between one of the five commercial and laboratory vaccine strains: F, ts-11, 6/85, K5831, K5054, and the challenge strain R low when tested on in vitro cultures. The assay K5831 vs. R low was also tested on specimens from live birds that were vaccinated with K5831 and challenged with R low, and successfully differentiated between the vaccine and the challenge strains in a quantitative manner. This preliminary in vivo application of the method also shed light on possible protection mechanisms for the M. gallisepticum K5831 vaccine strain.