The tr/// changes non-zero bytes to x. The while loop then uses index to search through the difference string for the x bytes and reports their index (0 based position).

I think it might be simpler to use a global regex match for non-nulls and pos. You can just match them for 1-based counting or put them in a look-ahead for 0-based counting. Note that I added another difference in the third characters to check that it was working as I had hoped.

use strict;
use warnings;

my $one  = "AGCTGATCGAGCTAGTACCCTAGCTC";
my $two  = "AGATGATCGAGCTAGTACCCTATCTC";
# Diffs here  ^                   ^

my $diff = $one ^ $two;

my @posns = ();

push @posns, pos $diff while $diff =~ m{[^\0]}g;
print qq{Differences, 1-based counting, at - @posns\n};

@posns = ();

push @posns, pos $diff while $diff =~ m{(?=[^\0])}g;
print qq{Differences, 0-based counting, at - @posns\n};
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The output.

Differences, 1-based counting, at - 3 23
Differences, 0-based counting, at - 2 22
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I hope this is of interest.

Cheers,

JohnGG