Mycoplasma gallisepticum (MG) can cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. Impacts of MG infections can include increased morbidity and mortality, decreased egg production, hatchability and feed efficiency. Biosecurity and bio-surveillance remain the primary means of deterrence as the current means of control are not wholly effective. Further applications towards the characterization of MG-related disease control requires proper understanding of the characteristics of MG infections with accurate and efficient quantification of in vivo MG populations. To this end, study was conducted to determine the in vivo MG populations in infected pullets and to determine impact of sampling schedule. The role of sampling in 3 distinct anatomical sites, and their associated MG populations were also compared. Results confirmed that sampling events did not affect MG populations and as a possible confounding factor, sampling frequency could be avoided for the future development of novel means of MG control.