You seem to misunderstand substr. The second argument is the start index to take the sub string from, the third argument the length.

If you have to take the first and third character of a string, you cannot do that with a single sub string, as you are not interested in the second character. Perhaps you want to do something along the lines of:

foreach my $tri (@trilet) {
    my ($f, $s, $t) = $tri =~ /(.)(.)(.)/;
    $first{"$f$s"}++;
    $second{"$s$t"}++;
    $third{"$f$t"}++;
}
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