Infections with Mycoplasma gallisepticum (MG) in poultry are associated with a wide range of disease conditions, including those affecting the respiratory and reproductive systems. The purpose of this study was to endorse the more sensitive diagnostic scheme for MG infection and identify the best molecular marker for MG phylogenetic analysis using six housekeeping genes: mgc2, mraW, atpG, ugpA, DUF31196, and lgT. For these purposes, 55 poultry flocks of different species were screened using either qRT-PCR or PCR techniques analogous to conventional culturing from non-cultured and cultured swabs on PPLO broth. The rate of MG positivity was the highest when using qRT-PCR from cultured broth (89.0%) and the lowest when using conventional culturing (34.5%). Compared to qRT-PCR from broth, statistical analysis using the Roc curve in MedCalc statistical software showed that the PCR schemes (qRT-PCR from swabs and PCR from swabs and broth) performed better than conventional culturing in terms of sensitivity, accuracy, and area under the curve (AUC), suggesting that they may be more reliable schemes. Further support was added by Cohen’s kappa test, showing moderate agreement between the molecular approaches. Among the six screened genes, mgc2 and mraW had the highest detection rates (69% and 65.4%, respectively). The comparative phylogenetic analysis revealed that mgc2 or atpG gene sequences distinguished MG isolates into different clades with high discriminatory power.