In February 2006, two outbreaks of highly pathogenic avian influenza A virus subtype H5N1 occurred in chickens in two neighboring districts (first in Nandurbar and second in Jalgaon) of Maharashtra, India, in a span of 12 days. In the present study, the neuraminidase (NA) gene of the two Indian H5N1 isolates was taken into consideration to find if the two strains are genetically similar. Phylogenetic analysis of the NA gene showed that the H5N1 strains isolated from the two outbreaks were not originated from the same source. The first Indian isolate (Nandubar/7972/06) was clustered closest to an isolate from chicken in Vietnam in 2004, whereas the second Indian isolate (Jalgaon/8824/06) showed resemblance to strains isolated from swan in Italy and Iran in 2006. Moreover, amino acid sequence analysis showed varying hot spots for substitutions between these two Indian isolates, and three substitutions were found at functional domain sites. Secondary structure changes due to these substitutions were also reported. This study reveals that the H5N1 strains isolated from chickens during 2006 bird flu outbreaks in two neighboring districts of Maharashtra, India are genetically different.