We compared the nucleotide sequences of the NS genes of 13 animal influenza viruses belonging to human, swine, avian, and equine viruses for the study of the genetic relatedness of the NS genes in animal influenza viruses. The NS genes of three virus strains A/chicken/Brescia/02, A/equine/Prague/56, and A/equine/Miami/63 were newly sequenced. The base sequence homologies between the NS genes of avian, human, swine, and the A/equine/Miami/63 viruses were 87.8% or higher. On the other hand, the base sequence of the NS gene of the A/equine/Prague/56 virus differed widely from those of other viruses analyzed in the present study. We constructed a model of the genetic tree of the NS genes of avian and equine influenza viruses by a modified Farris method. For comparison of the NS genes between human and avian viruses, we estimated the speed of the nucleotide substitutions of the avian influenza NS genes. It was roughly constant, even though the substitutions did not occur sequentially. The nucleotide substitution rate of the NS genes of avian influenza viruses was one-third to one-fourth that of human influenza viruses. We deduced the time of separation between the NS genes of human and avian influenza viruses during evolution.