Avian influenza is a highly infectious and dynamically evolving disease of birds causing high morbidity and mortality. It is caused by avian influenza virus (AIV) that belongs to the family Orthomyxoviridae. Two types of AIV have been described based on their pathogenicity viz. highly pathogenic avian influenza virus that causes severe disease with high mortality and low pathogenic avian influenza virus (LPAI) that generally causes asymptomatic infection or a mild disease. The H9N2 subtype is the widely circulated LPAI type in the world. The H9N2 subtype of was first reported from northern India in March 2003. However, systematical surveillance information for the evolution of H9N2 viruses in poultry flocks of Southern India is lacking. The present study reports the isolation and characterization of H9N2 isolates from the southern parts of the country during the period between May 2010 and September 2011. Out of the 30 poultry flocks investigated, six were found to be positive for HA activity. Further, all the six samples conformed as AIV. Partial nucleotide sequencing of the HA and NA genes revealed that all were belonging to the H9N2 subtype. Phylogenetically, the HA and NA genes of the H9N2 viruses from India clustered with those isolated from Bangladesh, Pakistan and the Middle East, although we were not able to conclude on their exact geographic origin.