Sero-conversion of pathogenic subtypes (H5, H7 and H9) of Avian Influenza Virus types was studied in commercial poultry farms in the Province of Punjab, Pakistan during the years 2015-16. Out of 6731 morbid tissues, only 11 samples (4 from broiler, 4 from broiler breeder and 3 from commercial layers farms) were found positive for Avian Influenza Virus (AIV) H9N2 type. Only one isolate was found positive for H9N2 subtype from 17994 tracheal swab samples. All the 37163 cloacal swab samples were found negative. The sero-analysis of 133438 blood sera samples showed sero-conversion in 66219 samples (49.64%) against H9 virus in the non-vaccinated poultry flocks, when tested with Heamagglutination Inhibition test (HI). Highest sero-conversion was recorded in broiler breeder (49.20%) followed by broilers (47.47%), layer (46.25%) and Native/Desi birds (45.59%). Highest HI titres were recorded in the flocks from Muzaffargarh, Rawalpindi, Toba Tek Singh, Sargodha, Gujrat, Faisalabad, Sheikhupura, Jhelum and Pakpattan while Bahawalpur, Gujranwala, Lahore, Jhang, Okara and Multan had relatively lower level of HI antibody titres. In conclusion only one type of AIV subtype H9N2 is prevailing in the province of the Punjab, Pakistan.