hash keys are unique, generally you do the reverse:

my %stuff = ();
while(<>){
        chomp;
        my @s = split /\t/,$_;
        my $r_name = $s[0];
        my $seq = $s[9];
        my $key = substr($seq,0,12);
        push @{$stuff{$key}}, $r_name;
}
# now %stuff is keyed on the value you wish to remain unique,
# which is easily determined because the array ref attached to
# it has a size of exactly 1 element
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