Seroprevalence and involvement of Mycoplasma gallisepticum (MG) in cases of chronic respiratory disease (CRD) in poultry and pheasantry birds of Northern Pakistan is underreported. In this study we report on the seroprevalence and isolation of MG from a total of 2341 poultry and 24 pheasantry birds. Overall, seropositivity of 46.56% (1090/2341) and 27.2% (6/24) against MG was observed among poultry- and pheasantry- birds, respectively. Broiler breeder and broiler showed 58.93% (264/448) and 37.23% (542/1456) seropositivity, respectively. A total of 17.1% (402/2341) samples collected from all these CRD-suspected birds were found positive on modified fray’s media indicating typical mycoplasma like colonies with appearance of fried egg or nipple like characteristic of 0.1 to 1 mm in diameter with a dense raised center in the middle. Interestingly, culture positivity for broiler samples was found higher (36.8%) as compared to breeders (33.5%) and layers (29.5%), respectively. Overall, of the cultured samples, 71.19% (215/302) were confirmed as M. gallisepticum by specie specific PCR. Furthermore, 82.43% of the confirmed isolates were recovered from Haripur, 82.35% recovered from Abbotabad and 60.53% were recovered from Mansehra. Finally, no significant difference (P<0.05) in PCR based positivity was found among all three types of bird population. Altogether, the observed high seropositivity and concomitant isolation of MG from CRD suspected birds investigated in the Northern region of Pakistan strongly suggest to initiate a comprehensive effective surveillance programme.