A couple of comments:

1) the open || die statement should actually be

open IN, $infile or die "Can't open input file: $!";
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For reasons of precedence.

2) Here are some faster algorithms. One way is to use a hash to record which characters you've already placed into your substitution string. Here's the main loop:

my %set;
my $substit = "";
for (1..26) {
    my $randchar;
    do { $randchar = chr((int rand 26) + 65) }
        while $set{$randchar}++;
    $substit .= $randchar;
}
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This way you don't have to do a search through the string each time.

And here's an even better way of doing it: build a random permutation of the alphabet string. To do this, we'll actually need an array:

my @alpha = split //, "ABCDEFGHIJKLMNOPQRSTUVWXYZ";
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Now we set another array equal to this one, then call the random shuffle algorithm on it:

my @crypt = @alpha;
fisher_yates_shuffle(\@crypt);
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which shuffles the array in place. Now all you need to do is get back the substitution string:

my $substit = join '', @crypt;
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Here's the definition of the fisher_yates_shuffle sub:

sub fisher_yates_shuffle {
    my $array = shift;
    for (my $i = @$array; --$i; ) {
        my $j = int rand ($i+1);
        next if $i == $j;
        @$array[$i, $j] = @$array[$j, $i];
    }
    return join '', @$array;
}
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(Taken directly from perlfaq4.)

I did some benchmarking on these, and here's what I got:

Benchmark: timing 5000 iterations of orig, f_yates, hash...
      orig: 33 secs (23.60 usr  0.00 sys = 23.60 cpu)
   f_yates:  5 secs ( 2.88 usr  0.00 sys =  2.88 cpu)
      hash: 13 secs ( 5.68 usr  0.00 sys =  5.68 cpu)
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where "orig" is the one you posted, "f_yates" is the one using fisher_yates_shuffle, and "hash" is the one using a hash to record seen characters.

If I've messed anything up, let me know.

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