I don't know if you have to have a hash of lists, but this could be solved using a hash of hashes. I couldn't find a .tsv file to try this out (but did try it on a .csv and it seemed to work).
while(<>){ chomp; my @s = split /\t/; my $r_name = $s[0]; my $seq = substr $s[9], 0, 12; $read2seq{$r_name}{$seq}++; } my @keys_with_2_unique_vals = grep 2 == keys %{$read2seq{$_}}, keys %read2seq;
UPDATE: To account for the change in OP's post. (He wants keys where both values are the same) Change the grep line to:

my @keys_with_equal_pair_of_vals = grep 1 == keys %{$read2seq{$_}}, keys %read2seq;

In reply to Re: extracting only those keys with uniq values in array list by Cristoforo
in thread extracting only those keys with uniq values in array list by v15

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