Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the two most important poultry pathogens as they cause huge economic losses in commercial poultry production. In the present study a duplex Polymerase Chain Reaction (PCR) assay targeting the Intergenic Spacer Region (IGSR, 16S-23S rRNA) of MG and 16S rRNA gene of MS was standardized for molecular detection of avian mycoplasmas directly from tissues of poultry affected with respiratory infections in Haryana. Ninety two pooled tissue samples including trachea, lungs and air sacs were collected from 92 different poultry flocks affected with respiratory infections and were subjected to mycoplasmological examination by duplex PCR. Result of duplex PCR revealed 25/92 (27%) MG, 2/92 (2.1%) MS and 1/92 (1.08%) samples were positive for both MG and MS. Conclusively, screening of flocks by duplex PCR could be helpful for surveillance and to recommend control measures to minimize prevalence and vertical transmission of avian mycoplasmosis.